UNIVERSITY OF HAWAl'I LIBRARY
DIVALENT CATION CHANNELS WITH INTRINSIC ALPHA-KINASE ACTIVITY
DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
BIOMEDICAL SCIENCES (PHYSIOLOGY)
MAY 2005
By Bret Fajans Bessac
Dissertation Committee: Andrea Fleig, Chairperson John R. Claybaugh David A. Lally Martin D. Rayner John G. Starkus Reinhold Penner ACKNOWLEDGEMENTS
The spirituality ofthis universe, thank you for the hope, mercy, love, passion,joy, grace, wonderment, perplexities, teachings, protection and companionship to my semi schizophrenic mind, dreams and seeking soul as my awareness, interpretation and enlightenment continues, as well as, the coral reefs, rain forests, beaches and sunshine, and their flora and fauna, ofthese islands.
My mother, Susanne Leppmann Bessac, MA, and my father, Francis Bagnall Bessac, PhD, thank you for the support, love, guidance, nurturing and shaping me with a stubborn, argumentative, never-surrender attitude, to perceive in the intellectual, humanistic, artistic and spiritual aspects oflife.
My love, Tricia Michelle Kratz, MSW, thank you for melting me with your heart, support, friendship and compassion, and for enduring and helping me through my studies and dissertation. Thank you for the friendship and ohana of your mother, Patricia Sonnenberg, and son, Matthew Kratz.
My advisor, Andrea Fleig, PhD, thank you for the patience, help, instruction, friendship, support, guidance and direction of my distractible wanderings in gathering comprehension of electrophysiology methods and the concepts of channels, as well as, knowledgeable daily discourse, insights in the experimental procedures, and keeping me on the direction and scope of my project. This dissertation would not have existed without you.
My committee member, Reinhold Penner, PhD/MD, thank you for your friendship and brilliant intellect, insights and discourse into science, electrophysiology, my project and hypothesis.
My committee member and graduate student advisor, David A. Lally, PhD, thank you for your friendship, help and support in getting through this process.
My committee member, John G. Starkus, PhD, thank you for your friendship, intrigue, insights and discourse in electrophysiology.
My committee member, Martin D. Rayner, PhD, thank you for your friendship, guidance, advise, support and setting the foundation for all this to occur.
My committee member, John R. Claybaugh, PhD, thank you for your friendship, intrigue, insight and discourse in physiology.
My sisters, brothers-in-law, nieces and nephews; Barbara Tracy, MS, MLT, David Tracy, Josh Tracy, Eli Tracy, Willow Tracy, Andrea Maxeiner, PhD, James Maxeiner, PhD/JD, Cassey Maxeiner, Peter Maxeiner, Turan Albini, Martin Albini, BSE, Fiona Albini, III Jethro Albini, Ivan Albini, Joan Steelquist, Mark Steelquist, Rueben Steelquist and Frances Steelquist thank you for all the love and support.
My editor and fellow laboratory mate, Andreas Beck, PhD, thank you for your friendship, humor, intellect, attention to detail and taking the time to read and re-read this dissertation and try to fonn it into something comprehensible.
Fellow graduate student and laboratory mate, Mahealani Monteilh-Zoller, MS, thank you for your friendship, humor, intellect and maintaining this laboratory.
Ka'ohi Dang and Carolyn Oki, MS, thank you for your friendship, cell culture and maintance ofthe laboratoqr.
My fellow laboratory mates, Phillip Demusse, PhD, Martin Kolisek, PhD, Henrique Chang, PhD/DVM thank you for your friendship, intellect and humor.
Helen Turner, PhD and Alexander James Stokes, PhD, thank you for your friendship, support and critical idea to help make logical sense from my muddy writings, as well as, techniques and insights in biotechnology and molecular biology.
The laboratory of Jean-Pierre Kinet in Boston, MA, thank you for the stably transfected mouse TRPM7 cells.
The laboratory of Andrew Scharenberg in Seattle, WA, thank you for the stably transfected human TRPM7, mutated human TRPM7 with the kinase not expressed and single-point mutated human TRPM7 of a glycine (1599) to a glutamate or a lysine to an arginine (1648).
The laboratory of Anne-Laure Perraud in Denver, CO, thank you for the stably transfected human TRPM6 or both TRPM6 and human TRPM7 or both TRPM6 and mutated kinase-deleted TRPM7.
The laboratory of Alexey Ryazanov in Piscataway, NJ, thank you for the stably transfected human TRPM7 with or without human annexin I or mutated annexin I of a serine to alanine or to aspartate.
The laboratory ofBernd Nilius in Brussels, Belgium, thank you for TRPM6 eDNA.
The constitution of the United States and Hawai'i and the taxpayers within these governments, as well as, The late Queen Ema, who paid for my dissertation from their institutions of National Institute of Health, University of Hawai'i and The Queen's Center for Biomedical Research.
IV ABSTRACT
TRPM7and its homologue TRPM6 are members of the melastatin-related Transient
Receptor-Potential (TRPM) family. TRPM6 and TRPM7 "chanzymes" form both transmembrane cation channels and cytosolic a -kinase enzymes. TRPM7 channel
2 2 specifically conducts Mg +, Ca + and trace divalent cations at resting potential, is
2 constitutively active and regulated by intracellular (IC) Mg + and Mg-ATP. TRPM7 a kinase phosphorylates TRPM7, annexin I and probably other proteins. TRPM7 is
2 2 ubiquitous, essential and implicated in cellular respiration and Mg + homeostasis, Ca + involvement in cell cycle and anoxic cell death.
TRPM7 over-expression induces HEK-293 cells to change morphology, detach and aggregate, but not immediately die. The properties of TRPM7 to induce these effects were examined for clues to TRPM7 physiological functions. TRPM7 channel was not
2 directly involved, because over-expression effects kinetics was similar in high Ca +, high
2 Mg + or standard media. Over-expression of TRPM7 without the kinase did not change cells, unless co-expressed with TRPM6. TRPM6 co-expression maintained kinase
2 2 deleted TRPM7 hypersensitivity to Mg +. Ca + free media mimicked the affects of
TRPM7 over-expression and increased the kinetics. TRPM7 kinase may bind and
2 inactivate a factor involved in cell morphology and adhesion, which involves Ca +.
Evidence shows it is not annexin-I.
TRPM7 osmosensitivity was examined, because osmotic stress signaling may involve membrane kinases and channels, and some TRP channels are osmosensitive. Hypertonic extracellular (EC) or hypo-osmotic IC media dose-dependently inhibits endogenous and v over-expressed TRPM7 currents in HEK-293 cells. Inhibition is not from capacitance or
2 ionic strength changes, and is independent of f-actin, cAMP, pH, Mg-ATP or Mg +.
Countering osmotic pressure with pressure through the patch-pipette cancels hypertonic inhibition of TRPM7. Hypotonic EC application rapidly increases over-expressed
2 TRPM7 currents suppressed with IC Mg + (3 mM), Mg-ATP (4 mM), hypo-osmolar IC media or a-kinase deletion, but does not affect endogenous TRPM7. Hyper-osmotic IC media also facilitates TRPM7. It was found TRPM7 is not involved in osmotic stress
2 induced volume regulation. Possibly IC Mg + fluctuations or mechanosensitivity explain
TRPM7osmosensitivity.
TRPM7 channel osmosensitivity and TRPM7 kinase apparent binding factors of morphology and adhesion suggest new possible physiological functions of TRPM7 of initiating signaling cascades and regulating proteins involved in adjusting cellular morphology, membrane dynamics or growth.
vi TABLE OF CONTENTS
ACKNOWLEDGMENTS .iii-iv
ABSTRACT v-vi
LIST OF TABLES x
LIST OF FIGURES xi-xii
LIST OF ABBREVIATIONS xiii-xvi
CHAPTER 1: INTRODUCTION 1-18
1.1. TRPM6 & TRPM7: Divalent Cation Channels with a-Kinases 1-5
1.2. Glutamate Effect on TRPM7 Pore and Divalent Cation Permeation 5-7
1.3. The Channel and the Kinase Involvement in TRPM7 Over-Expression-
Induced-Changes ofCell Morphology 8-14
1.3.1. TRPM6, TRPM7 and TRPM6/7: Physiological Roles ofChannels 8-10
1.3.2. TRPM6 and TRPM7: Physiological Roles ofthe a-kinase 10-14
104. TRPM6 and TRPM7 Osmosensitivity 14-18
1.5. Synopsis ofObjectives 18
CHAPTER 2: MOLECULAR CHARACTERISTICS 19-36
2.1. Transient Receptor Potential (TRP) Channels 19-25
2.1.1. Transient Receptor Potential (TRP) Channels 19-22
2.1.2. Melastatin-like Transient Receptor (TRPM) Channels 23-25
2.2. TRPM6 and TRPM7 Primary Structure 25-36
2.2.1. PROSCAN Putative Functional Motifs 25-27
2.2.2. Amino-terminal Region ofTRPM6 and TRPM7 28-29
Vll 2.2.3. Putative Transmembrane Region ofTRPM6 and TRPM7 .29-33
2.2.4. Carboxyl-terminal Region ofTRPM6 and TRPM7 .33-36
CHAPTER 3: MATERIALS AND METHODS 37-51
3.1. Cells 37-39
3.2. Solutions 39-40
3.3. Patch-Clamp Electrophysiology .41-44
3.3.1. Whole-cell Patch-clamp Method .41-43
3.3.2. Solution Application 44
3.3.3. "Balloon" Patch Volume Application .44
3.4. Fura-2: Fluorescent Indicator of Calcium and Cell Volume .45-48
3.5. Cell Vitality Staining and Microscopy .49
CHAPTER 4: RESULTS 50-78
4.1. TRPM7 Cell Volume-Membrane Strain Sensitivity 50-56
4.1.1. Hypertonic-induced Inhibition ofTRPM7 and MagNuM 50-56
4.1.2. Hypotonic-induced facilitation ofTRPM7 56-58
4.1.3. Balloon Patch Counteracts Hypertonic Inhibition ofTRPM7 59-60
4.1.4. Cytochalasin D, pH nor cAMP Alter Osmotic Effects on TRPM7 60-61
4.1.5. Intracellular Osmotic Stress Effects on TRPM7 Currents 61-63
4.] .6. Truncated TRPM7 without the Kinase Domain is Hyper-sensitive to
Osmotic Stressors 64-65
4.1.7. TRPM6/7 Osmotic Sensitivities 65-67
Vlll 4.1.8. TRPM7 Over-Expression Does Not Alter Cell Volume-Responses to
Osmolarity 67-68
4.2. TRPM7 and TRPM6/7 Over-Expression Effects 69-77
4.2.1. TRPM7 And TRPM6/7 Over-Expression Effects 69-71
2 2 4.2.2. Ca + and Mg + Media Levels on TRPM7 Over-Expression Effects 71-73
4.2.3. Kinase Mutations Effects on Cells Adhesion and Morphology 73-74
4.2.4. Annexin-I Co-Expression on TRPM7 Over-Expression Effects 74-76
4.2.5. Electrophysiological Currents ofOver-Expressing Cells 76-79
4.3. Glutamate Facilitates Outward, But Not Inward TRPM7 Currents 79-82
CHAPTER 5: DISCUSSION 83-101
5.1. TRPM7 Channel Osmosensitivity 85-88
2 5.1.1. Hypotonic Facilitation ofTRPM7 as an Intracellular Ca + Signal.. 85
5.1.2. Intracellular Signaling ofHypertonic Stress 86
5.1.3. Gating-Mechanism 86-92
5.2. TRPM7 a-Kinase is Involved in Cell Morphology and Adhesion 93-98
5.3. Extracellular Sodium Glutamate Effects on TRPM7 99-100
5.4. Summary 100-101
REFERENCES 102-113
APPENDIX I: TRPM AMINO ACID SEQUENCE ALIGNMENT a-e
APPENDIX II: TRPM6 & TRPM7 ALIGNMENT WITH MOTIFS f-h
APPENDIX III: DEFINITION OF MOTIFS FROM PROSITE DATABASE BY
THE SWISS INSTITUTE OF BIOINFORMATICS .i-pp
IX LIST OF TABLES
Table Page
2.1. TRPM sequences alignment identically or similarly 25
2.2. PROSCAN functional motifs in TRPM6 or TRPM7 27
x LIST OF FIGURES
Figure Page
2.1. Human, C. elegans, rat TRPC2, the original Drosophila TRP and TRP-like proteins ofyeast E. coli and Leishmania TRP protein sequence similarity 20
2.2. Human TRP (& rat TRPC2) proteins sequence similarity 21
2.3. Human TRPM proteins sequence similarity 24
2.4. TRPM6 and TRPM7 amino acid sequence alignment for the N-terminal span ...27
2.5. Streptomyces K+ channel structure surmised by X-ray crystallography 29
2.6. Transmembrane, ion pore and conserved C-terminal span to coiled-coil region..30
2.7. Putative alignments ofTRPM channels cation selectivity filter and pore region.31
2.8. The putative sixth transmembrane domain of TRPM channels and a portion of an extracellular loop alignment ofamino acids .31
2.9. TRPM7 a-kinase domain secondary structure from X-ray crystallography 33
2.10. TRPM6 and TRPM7 a-kinase and C-terminal amino acid sequence alignmenL34
2.11. Unique TRPM6 and TRPM7 amino acid sequence span 34
3.1. The intensity of 512 nm light emitted by a concentration of Fura-2 with various 2 Ca + concentrations in response to light wavelengths from 250 nm to 450 nm.. .46
4.1.1. Hypertonic conditions inhibit the endogenous TRPM7 currents (MagNuM) .....51
4.1.2. Hypertonic conditions inhibit TRPM7 52-53
4.1.3. TRPM7 inhibition by hyperosmolarity is in a dose dependent manner.. 53
2 4.1.4. Hypertonic conditions simultaneously inhibit TRPM7 currents and Ca + influx.. 54
4.1.5. Hypertonic inhibition ofTRPM7 occurs in nominal divalent cation conditions, but is blunted in conditions devoid ofdivalent cations 55-56
2 4.1.6. Hypotonic solutions facilitate TRPM7 partially inhibited by Mg + or Mg·ATP...58
Xl 4.1.7. Balloon patch rapidly reverses hypertonic inhibition 60
4.1.8. Osmotic effects on TRPM7are not altered by pH or cAMP or cytochalasin D....61
4.1.9. Hypo-osmotic or hyper-osmotic intracellular conditions effect TRPM7 and the effects are removed with an equal osmotic extracellular application 63
4.1.10. TRPM7 with the kinase domain deleted is hypersensitive to osmotic stress 65
4.1.11. Osmotic stress effects occur on currents carried by TRPM6 co-transfected with TRPM7 with or without the kinase domain 66-67
4.1.12. TRPM7 over-expression dose not alter cell volume regulation to osmotic stress.68
4.2.1. Over-expression of TRPM7 or both TRPM6 and TRPM7 induces cells to round, swell and detach, in a time-dependent manner 70
4.2.2. TRPM7 over-expression does not induce cells to die, but to detach, which can be partly rescued by coating the cover slips with poly-lysine 71
4.2.3. TRPM7 over-expressing effects are similar and additive to cells incubated in 2 2 2 Ca +- free media, while high Ca + or Mg + media has little or no effect 72
4.2.4. Over-expression of TRPM7 without a kinase does not induce any noticeable morphological changes unless co-expressed of TRPM6 or with the kinase only disabled, but remaining structurally intact. 74
4.2.5. TRPM7 over-expression effects are not affected by annexin I co-expression.....75
4.2.6. The I-V plots of TRPM617 and TRPM7 are identical, as are the I-V plots of TRPM6 and non-induced cells 77
2 4.2.7. Kinase-deleted mutant of TRPM7 currents are hyper-sensitive to Mg + with or without co-expression ofTRPM6 79
4.3.1. Extracellular sodium glutamate facilitates TRPM7 outward currents 80
4.3.2. Extracellular sodium glutamate facilitation of TRPM7 outward currents is due to divalent cation conductance, not ion exclusion or channel open probability 81
4.3.3. Extracellular hypertonic inhibition and sodium glutamate facilitation of TRPM7 carried currents occur simultaneous and independent 82
XII LIST OF ABBREVIATIONS
ADPR: adenosine diphosphoribose
ATP, ADP and AMP: adenosine tri-, di- and mono-phosphate
BAPTA: 1,2-Bis (2-Aminophenoxy)Ethane N,N,N',N'-tetraacetic Acid
Ca,2+ Mg,2+ Mn,2+ Zn2+ , C02+ , Na, + Cs +an d K+ : catIOns. 0 fl·ca ClUm, magnesIUm,. manganese, zinc, cobalt, sodium, cesium and potassium, respectively
[Ca2+h and [Mg2+h : intracellular calcium and magnesium concentration cAMP: cyclic adenosine mono-phosphate cGMP: cyclic guanosine mono-phosphate cr: chloride anion
CPK: conventional protein kinases
C-t: carboxyl-terminal span
DC: direct current
DCT: distal convoluted tubule
DIDS: 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid
DMEM: Dulbecco's modified Eagle medium
DMSO: dimethylsulfoxide
DNA and cDNA: deoxy-ribonucleic acid and clonal deoxy-ribonucleic acid
DVF+EDTA: divalent cation free with 5 mM Na2EDTA
DVF: divalent cation free
ECF: extracellular fluid
EDTA: Ethylenediaminetetraacetate
Xlll g, mg and Ilg: grams, milligrams and micrograms mass
G1599E and K1648R: single-point mutated human TRPM7 ofthe a-kinase region glycine or lysine
GFP: green fluorescent protein
HEDTA: hydroxyethylenediarninetriacetic acid
HEK-293: Human embryo kidney fibroblasts capable ofseveral passages
HOG: high osmotic glycerol hTRPM7: recombinant human TRPM7
IPJ;' inositol 1,4,5-tris-phosphate
I-V: current-voltage kHz: kilohertz
1, ml and Ill: liters, milliliters and microliters
M, mM and 11M: molar, millimolar and micromolar concentrations
MagNuM: Magnesium Nucleotide Metal, the endogenous TRPM7-associated current min, s, ms and IlS: minutes, seconds, milliseconds and microseconds mOsm: milli-osmoles mRNA: messenger ribonucleic acid mV: milliVolts mV/pA: milliVolts/ picoAmpere mTRPM7: recombinant mouse TRPM7
MQ and GQ: Mega-ohms and Giga-ohms n: number
XIV nA and pA: nano- and pico- Amperes
NAD: nicotinamide-adenine dinucleotide
NaGlu: sodium glutamate
NaOH: sodium hydroxide nm: nanometers no-tet-HEK: HEK-293s without tetracycline-induction ofTetR
N-t: amino-terminal span
N-terminal: amino-terminal pH: log hydronium cation (proton) concentration
PIP2: phosphatidylinositol 4,5-bisphosphate siRNA: small interfering ribonucleic acid
TetO: tetracycline-operator
TetR: tetracycline repressor protein
TRP: Transient receptor potential (protein or channel)
TRPA or ANKTM: TRP protein ankyrin-transmembrane protein I
TRPC and TRPCl, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6: Members ofthe TRP canonical protein sub-family
TM: transmembrane domains
TRPM and TRPMl, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8: Members ofthe TRP melastatin protein sub-family
TRPM617: co-expressed and putative heteromer ofTRPM6 and TRPM7
TRPM6-GFP: transiently transfected human TRPM6
TRPM6+M7-~-kinase:co-expression ofhuman TRPM6 with TRPM7-~-kinase
xv TRPM7+M6-GFP: transiently transfected human TRPM6 co-expressed with stably transfected human TRPM7
TRPM7-~-kinase: human TRPM7 truncation mutant lacking the carboxyl-terminal region including the a-kinase
TRPMLI and TRPML2: mucolipin TRP proteins
TRPN or NOMC: non-mechanosensation channel TRP protein
TRPP and TRPPl, TRPP2, TRPP3: polycystin TRP proteins
TRPV and TRPVl, TRPV2, TRPV3, TRPV4, TRPV5, TRPV6: Members ofthe TRP vanilloid protein sub-family
TyrKP: tyrosine kinase phosphorylation
UV: ultraviolet wt-HEK: HEK-293s without vector ofchannel, TetR and TetO
't': time constant
XVI CHAPTER 1. INTRODUCTION
1.1. TRPM6 & TRPM7: DIVALENT CATION CHANNELS WITH a-KINASES
The vibrant green Kipahulu canopy, the grey bustling Honolulu freeways, the silent depths of black coral forests, glowing crimson ooze of Kilauea lapahoehoe and pearly smiles of Hawai'i, all contain magnesium and calcium. These alkaline earth metal divalent cations are also required for the vision of these images, their incorporation into memories, stabilizing the DNA involved in our unique perspective and most of our body processes. Elemental Mg and Ca readily lose 2 electrons, resulting in an excess of two protons and a charge of2+.
2 The adult body has about 30 g Mg and 1300 g Ca with more than half of the Mg +
2 and 99% of the Ca + in the bone as hydroxyapatite crystals. In the extracellular fluid
2 2 Mg + and Ca + are similarly concentrated, as free aqueous ions and in complex with plasma proteins, such as albumin, and anions, such as citrate and phosphate (0.75 - 1.05 mM [Mg2+]; 2.1 - 2.6 mM [Ca2+]). Insufficient plasma Ca2+ or Mg2+ increases parathyroid
2 2 hormone, which stimulates release of Ca + and Mg + from the bones and enhances intestinal absorption and renal reabsorption ofboth cations.
2 Inside cells, Mg + is considerably the most abundant free divalent cation. A fraction of intracellular Mg2+ ([Mg2+]i) is in the soluble "free" form (0.5 - 1.0 mM), while the remainder is primarily bound to the phosphate and carboxyl moieties of proteins, lipids
2 and nucleotides. Nearly every molecule and function in the cell appears to involve Mg +.
2 Mg + stabilizes the phosphate head groups of the lipid membranes and the nucleotide phosphoesters of free-nucleotide tri- and di- phosphates and those bound within the DNA 2 backbone. Many protein functions are regulated or modulated by Mg2+or Mg- ATP, such as actin, myosin, ~-adrenergic receptor-adenylate cyclase complex, ATPases and certain
K+ and Ca2+ channels. In many enzymes, Mg2+ is the catalytic metal co-factor for the enzymatic reactions (for review see the report by the UN WHO and FAO, 2002).
The cytosol is kept principally devoid of Ca2+ (less than 0.1 mM) by chelating proteins and transportation into intracellular stores, such as the endoplasmic reticulum.
This allows cells to use transient boluses of Ca2+ from these intracellular stores and or from the extracellular fluid to initiate cellular events. Ca2+ directly or in complex with calmodulin binds to certain proteins changing their structure, which in tum initiates or regulates the actions of these proteins, such as those proteins involved in apoptotic cascades, neuronal plasticity, vision, memories, cell differentiation, migration, cell cycle, muscle contractions and exocytosis (for review ofCa2+in cell physiology see Dedman &
Kaetzel, 2001 and Nemeth, 2001).
Ion channels and other specialized proteins allow Mg2+ and Ca2+ to move past the non-polar barriers of cell plasma membranes from the extracellular fluid into the cytosol and vice versa. The opening and closing of the ion channels and consequent ionic flux through the channels are regulated by stimuli specific to the particular channel. Ions enter into and through open channels by random diffusion and by electromagnetic attraction and repulsion from other charges, both, inside and outside of the cell, and to a smaller extent, the amino acid residues within or near the ion pore (for review see Hille, 2001).
The electrical properties of ions allow for the channel ion conductance and overall ionic flux to be measured with electrophysiological techniques. Selective fluorescent indicators are also used to monitor intracellular concentrations of Ca2+ and, to certain 3 2 2 extent, Mg +. Many channels have been characterized for Ca + by these techniques. Yet,
2 the only mammalian channels found to be involved with conducting Mg + across the plasma membrane are TRPM6, TRPM7, and their heteromer (Nadler et aI., 2001;
Chubanov et aI., 2004; Voets et aI., 2004). These proteins are designated "Chanzymes"
(channel + enzymes), for their ability to form both transmembrane Transient Receptor
Potential (TRP) channels and intrinsic cytosolic a-kinases (Nadler et aI., 2001; Runnels
et aI., 200I, Yamaguchi et a., 2001). In chapter 2 TRP channels and the molecular
characteristics ofTRPM6 and TRPM7 proteins are more thoroughly discussed.
2 2 Mg + charge is slightly larger than Ca + and much larger than ions with just a 1+
charge, such as Na+ or K+. The larger the charge of an ion, the stronger the
electromagnetic force will be with other charges. In the aqueous environment of the body, Mg2+ attracts partially negatively charged oxygens of water to surround it in a
2 2 hydration-shell. Mg +,s large charge allows for Mg + to attract more water molecules than most aqueous ions, making the hydrated-Mg2+ a larger size than the other hydrated-ions.
At negative membrane potentials, TRPM6 and TRPM7 specifically carry divalent cations
into the cell, while at positive potentials the channels carry an efflux of primarily
monovalent cations (Nadler et aI., 2001). The mechanism that these channels exclude the
smaller cations at negative potentials, but not at positive potentials, is theorized to be due
to a divalent cation permeation block. At small positive potentials, a divalent cation
permeation block appears to exists in some channels. In order to further understand the
divalent cation permeation block, the permeation block at small positive potentials was
investigated. 4 Many characteristics and insights into the physiological roles of TRPM7 have been discovered, while data on TRPM6 is more limited. These include insights in: TRPM7 channel divalent cation conductance and divalent cation permeation block; difference of
2 2 TRPM7 from store operated Ca + channels; Mg +, Mg" ATP, cAMP, PIP2 and pH
2 regulation of TRPM7; Mg + regulation of TRPM6; X-ray crystallography of TRPM7 a-kinase; a-kinase and divalent cation interactions; a-kinase auto-phosphorylation of
TRPM7 and phosphorylation of annexin I; heteromer formation of TRPM6 and TRPM7; mutations of TRPM6 linked to genetic hypomagnesemia; TRPM6 immunocytochemical
2 staining in the kidney; and TRPMTs physiological and pathophysiological roles in Mg +
2 2 homeostasis, Ca + regulation of cell cycle and Ca + induced excitotoxic protected anoxic neuronal death (Nadler et aI., 2001; Schmitz et aI., 2004; Aarts et aI., 2003; Hanano et aI,
2004; Dorovkov & Ryazanov, 2004; Walder et aI., 1997; Schlingmann et aI, 2002;
Walder et aI.,2002; Voets et aI., 2004; Chubanov et aI., 2004; Ryazanov et aI., 1999;
Yamaguchi et aI., 2001; Runnels et aI., 2001; Ryazanova et aI., 2004; Monteilh-Zoller et aI., 2003; Hermosura et aI., 2002; Kozak et aI., 2003; Gwanyanya et aI., 2004; Runnels et aI., 2002; Takezawa et aI., 2004; Kerschbaum et aI., 2003). However, the physiological functions and roles of TRPM6 and TRPM7 channel divalent cation transport and kinase phosphotransfer activities and the purpose of the two existing in the same protein are not well understood.
2 Cells without TRPM7 protein expression die, unless incubated in high Mg + (Schmitz et aI., 2004). Too much expression of recombinant TRPM7 leads cells to become round, swell, clump and detach (Nadler et aI., 2001). The properties of TRPM7 responsible for the effects on cell morphology and adhesion are not known, but many explanations 5 suggest the involvement of TRPM7 channel divalent cation conductance and or TRPM7 a-kinase activity. Therefore, the channel and the kinase involvement in inducing TRPM7 over-expression effects were examined.
Osmotic stress signaling in yeast and invertebrates involves plasma membrane kinases and channels (Hohmann, 2002; Colbert et aI., 1997; Solomon et aI., 2004).
TRPM7 and TRPM6 contain both. Other TRP channels' are osmosensitive and TRPM6 is expressed in cells likely experiencing steep transcellular osmotic gradients (Schlingmann et aI., 2002; Voets et aI., 2004). Therefore, the possibility of TRPM7 and or TRPM6 having a novel physiological role and characteristic of sensing osmolarity was explored.
1.2. GLUTAMATE EFFECT ON TRPM7 PORE AND DIVALENT CATION
PERMEATION
At negative potentials, such as cell resting potential, open TRPM6, TRPM7 and putatively TRPM617 channels specifically conduct divalent cations into the cell (Nadler et aI., 2001; Monteilh-Zoller et aI., 2003; Voets et aI., 2004). The specificity of an ion channel is by the protein(s) forming a selectivity filter. The proteins of Na+ and K+ channels use slight differences in charge and the size of the hydration shell encases K+ and Na+ to specifically filter one ion over the other. A key feature of this filter is the spacing of a ring of pedtidyl amide oxygens of glycines coupled to an aromatic residue
(one from each tetramer). The oxygens mimic the hydration shell around the specific ion and strip offthe surrounding waters. Ifthe ion is too big or small, the protein oxygens fail to line-up with the hydration shell oxygens, thus the hydration shell cannot be stripped. 6 The hydrated ion is too large to move into the channel pore region (For review on selectivity filters and ion pore theory see Hille, 2001).
There is strong evidence that TRPM7 stripping off of the hydration shell is not specific for a particular cation, instead divalent cation specificity may arise from a divalent cation permeation block ofthe channel by a series ofdivalent cations in the pore.
The theory is that the open TRPM7 negatively charged water-stripping pore interaction with divalent cations is so strong that a divalent cation becomes trapped in the pore.
When another divalent cation enters the channel, the repulsive force of the entering divalent releases the trapped divalent cation into the cell and the entering divalent cation becomes trapped in the channel. When a monovalent cation attempts to enter the channel, it does not have enough charge to form a strong enough repulsive force to dislodge the trapped divalent cation, therefore it cannot go into the channel. The kinetics of the channel block by extracellular organic polyvalent cations suggests divalent cations bind at a series of sites within the cation pore forming a train of divalent cations to push the most forward divalent cation into the cell (Kerschbaum et aI., 2003).
The divalent cation permeation block can be from either intracellular or extracellular divalent cation. The block still occurs, when one side contains divalent cation, while the other is maintained free of divalent cations. However, the permeation block occurs at potentials associated with divalent cation influx from extracellular fluid. As potentials become increasingly positive, TRPM6 and TRPM7 channels lose their divalent cation permeation block and carry a linear current ofprimarily Cs+ or K+ efflux (depending on internal media). When both solutions are free of divalent cations, then there are no divalent cations to clog the channel pore, thus monovalent cations are allowed to freely 7 flow through TRPM7 in a linear voltage-current relationship at both positive and negative
potentials (Nadler et aI., 2001).
Unidirectional divalent cation-conductance may explain why, intracellular polyvalent
cations do not block the channel, the lack of divalent cation permeation block at more
2 positive potentials, and the similar or greater ability of TRPM7 to conduct Mg + into the
cell even though the concentration gradient is much greater for ci+ (Monteilh-Zolleret
aI., 2003; Kerschbaum et aI., 2003). Unidirectional divalent cation conductance would
only be determined by the extracellular fluid (ECF).concentration of the divalent cation
and the forces acting on bringing the divalent cation to and through the channel. These
forces would be the channel amino acid residue negative charges and the electric field
from the relatively negatively charged intracellular side ofthe membrane (Hille, 200 I).
The hypothesis that the negatively charged amino acid residues in the pore attract
divalent cations into the channel at positive potentials was examined
electrophysiologically using the net negatively charged amino acid glutamate as the
extracellular anion instead of Cr. According to the above hypothesis, the channel's negative residues could potentially be sequestered by glutamate and only negative potentials would attract divalent cations into the channel, while at positive potentials, the
channel would carry a linear monovalent efflux. 8 1.3. THE CHANNEL AND THE KINASE INVOLVEMENT IN TRPM7 OVER-
EXPRESSION-INDUCED CHANGES OF CELL MORPHOLOGY
1.3.1. TRPM6, TRPM7 and TRPM6/7: Physiological Roles ofthe Channels
Evidence clearly suggests TRPM6 and TRPM7 have physiological functions in the
2 transport of Mg + from the extracellular fluid to the cytosol. TRPM6, TRPM7 and
2 TRPM6/7 are implicated in the duodenal and ileal absorption ofMg + (~200 mg/day) and
2 2 in distal convoluted tubulular (DCT) Mg + reabsorption (5-10% of total Mg + reabsorption) (Greger et aI., 1981; Fine et aI., 1991; Walder et aI., 2002; Schlingmann et
2 aI., 2002; Voets et aI., 2004; Chubanov et aI., 2004; for review of Mg + reabsorption see
Satoh & Romero, 2002 and Meij et aI., 2002). TRPM6 mRNA is translated in the intestine and kidney cells and TRPM6 protein expression in the kidney is exclusively in the DCT (Schlingmann et aI., 2002; Voets et aI., 2004). In the physiological range of 0.5
2 to 1.5 mM, intracellular Mg + negatively regulates TRPM6 channel activity. Humans and
2 2 animals with low serum Mg + have increased intestinal Mg + absorption, which could be
2 explained by TRPM6 and its regulation by Mg + (Meij et aI., 2002; Voets et aI., 2004).
Certain genetic hypomagnesemias have been linked to mutations ofTRPM6 (Walder
et aI., 1997; Walder et aI., 2002; Schlingmann et aI., 2002). Many other pathological
conditions and symptoms are associated with hypomagnesemia and most are due to low
[Mg2+]i, the most dangerous being cardioinfarct (http://www.mgwatcr.com/index.shtml
provides discussions and a list ofvarious sites and publications on Mg pathology).
2 Cellular absorption of Mg + from the extracellular fluid to the cytosol appears to
involve TRPM7 and this role is vital and ubiquitous (Nadler et aI., 2001; Schmitz et aI.,
2004). TRPM7 knockout cells stop proliferating and die, unless these cells are incubated 9 2 III 10 mM Mg + (Schmitz et aI., 2004). Similar to TRPM6, physiological intracellular
2 concentrations of Mg + negatively regulate TRPM7 and intracellular dialysis of media
2 containing low or no Mg + increase the open probability of the channel (Nadler et aI.,
200 I; Kerschbaum et aI., 2002).
2 Even though, Mg + is the predominant free divalent cation in the cell, little is known
2 about the effects of chronic high intracellular Mg +. Perhaps, over-expression ofTRPM7
2 upsets Mg + homeostasis and the over-expression effects are the result overloading the
2 cells with Mg + due to the increased number of channels expressed. However, this
2 explanation does not address that incubation of cells in high Mg + containing media does
2 not necessarily increase intracellular Mg + and TRPM7 is tightly regulated by
physiological [Mg2+]i (Nadler et aI., 2001; for review see Wolfet aI., 2003).
2 TRPM7 also conducts Ca + and other trace divalent cations in manner similar to its
2 Mg + conduction (Monteilh-Zoller et aI., 2003). Increased TRPM7 channel activity is
shown to increase intracellular Ca2+ concentration ([Ca2+]i), while TRPM7 knock-down
with siRNA, decreases [Ci+]i (Monteilh-Zoller et aI., 2003; Hanano et aI., 2004). [Ca2+]i
can negatively regulate TRPM7, but the influence is small due to the physiological low
level of [Ca2+]i (Monteilh-Zoller et aI., 2003). High intracellular Ca2+ induces a plethora
of changes in cell physiology, including swelling, granulation, blebbing, and apoptosis
(for review see Dedman & Kaetzel, 2001). Possibly, TRPM7 over-expression effects are
2 from high intracellular Ca + due to TRPM7 channel over-expression.
TRPM7 channel may playa role in cell respiration and cell death from starvation.
Cellular respiration causes [Mg2+]i and [Ca2+]i to fluctuate with changes in Mg'ATP and
Mg'ADP concentrations. The changes in [Ca2+]i regulate the Ca2+-facilitated 10 glycogenolytic and glycolytic cascades. Mg-ATP negatively regulates TRPM7, thus depletion of intracellular Mg'ATP results in TRPM7 channels becoming more active, resulting in increased [Mg2+]i and [Ca2+]i (Nadler et aI., 2001, Monteilh-Zoller et aI.,
2003; Hermosura et aI., 2002). Anoxic high [Ca2+Ji-induced cell death can be prevented by large divalent ions or other blockers of Ca2+ and Mg2+ channels. Excitotoxic independent anoxic Ca2+ increase and subsequent cortical neuronal death can be protected by TRPM7 siRNA knock-down (Aarts et aI., 2003). Anoxia lowers cell pH, which inhibits TRPM7 or TRPM617 heteromer and may be protective to anoxic-induced cell death (Kozak et aI., 2004; Gwanyanya et aI., 2004). TRPM7 over-expression and subsequent increase of intracellular Mg2+ and Ca2+ may be inducing changes in cells that mimic the cell morphology changes and necrosis of the over-active endogenous TRPM7 in anoxic conditions. Therefore, TRPM7 over-expressing cells vitality was measured.
1.3.2. TRPM7 and TRPM6: Physiological Roles ofthe a-kinase
In addition to forming channels TRPM6 and TRPM7 form cytosolic a-kinases. These kinases transfer a phosphate from ATP to a target protein serine or threonine hydroxyl moiety, but unlike the conventional serine/threonine kinases, these phosphorylations are within an a-helical secondary structure of the target protein. TRPM7 kinase auto phosphorylation of threonines in TRPM7 protein is enhanced by Mg2+ and Mn2+, inhibited by Zn2+and Co2+, but not affected by Ca2+(Ryazanova et aI., 2004). The kinase likely is in close proximity to the cytosolic side of TRPM7 pore divalent cation influx
(Yamaguchi et aI., 200I). A physiological role ofthe channel divalent cation conductance might be to regulate a-kinase activity, which in tum regulates TRPM7 and other proteins. 11 Therefore, the kinase may be ultimately responsible for TRPM7 over-expression effects, but be dependent upon the channel activity.
2 Another scenario is that, the kinase and its regulation by Mg + might be involved in
2 adjusting the channel Mg + conductance, either directly or indirectly by adjusting channel
2 2 sensitivity to Mg +. Intracellular Mg + negative regulation of channel activity has been shown to be enhanced by removal of the kinase, but does not differ or is blunted with single-point mutations that disrupt kinase function but not the protein structure (Schmitz et aI., 2004). This implies that the loss of the kinase and not loss of the catalytic site or the target protein binding site, physically alters TRPM7 protein properties. Thus, the
2 mechanism within TRPM7 protein structure involved in Mg + inhibition becomes hypersensitive. Another possibility is that the kinase associates with a protein involved in
2 modulating the Mg + effects. TRPM7 over-expression effects might be through the over
2 expressed kinase removing the channel sensitivity to Mg + and thereby, permitting the
2 channel to over-load the cell with Mg +.
In addition to the a-kinase auto-phosphorylating TRPM7, the a-kinase has been shown to phosphorylate a conserved serine in an N-terminal a-helix of annexin I protein
(Runnels et aI., 2001; Yamaguchi et aI., 2001; Dorovkov et aI., 2004; Schmitz et aI.,
2 2004). In order to be activated, annexin I must undergo a Ca +-dependent conformational change to expose the serine for phosphorylation (for review Gerke & Moss, 2002). The
2 2 role of Ca + on annexin I, explains why increases in Ca +, increase TRPM7-kinase phosphorylation of annexin I, but not auto-phosphorylation of TRPM7 (Dorovkov et aI.,
2004; Ryazanova et aI., 2004). Pharmacological studies suggest the phosphorylation of annexin I serine involves kinases, such as protein kinase C, IP3-kinase and MAP kinase 12 (Solito et al., 2003). However, the pharmacological agents might be disrupting TRPM7 a-kinase activity, since TRPM7 a-kinase pharmacological characteristics are not known.
Also known as lipocortin I, annexin I mediates many of the regulatory effects of glucocorticoids, including roles in arachidonic acid release, cell growth, inflammation, ischemic damage, pain, release ofneuropeptides, to name a few. Glucocorticoids regulate the expression of annexin I and translocate it from the cytosol to the outer surface of the plasma membrane in the active form with the serine phosphorylated (Solito et al., 2003).
Annexin I knock-out mice are relatively healthy and reproductive, but display a loss of many ofthe actions ofglucocorticoids on regulating the immune system (Roviezzo et aI.,
2002). The interaction and possible regulation ofannexin I by TRPM7 has many exciting possibilities for future novel pharmacological targets as well as, comprehension of diseases associated with glucocorticoid actions in the nervous system (anxiety, depression, neurodegeneration, sleep cycle, pain...), the immune system (allergies, asthma, skin disorders, arthritis, Crohn's disease...), the metabolic system, and other systems ofthe body (for review see Roviezzo et aI., 2002 and John et aI., 2004). Annexin
II glucocorticoid physiological actions often involve changes in cell morphology, adhesion and aggregation and over-activity or inactivation of annexin I by TRPM7 might be the cause ofTRPM7 over-expression effects.
In the amoeba, Dictyostelium, a group of a-kinases phosphoregulate the a-helical tails ofmyosin proteins (for review see De La Roche & Cote, 2001). By analogy, TRPM7 a-kinase has been suggested to regulate myosins in mammalian cells (Langeslang et aI.,
2004). Cortical annexin, myosins and actin interrelate and independently have actions on cell adhesion, migration, volume regulation, plasma membrane tension and muscle 13 2 2 contraction. TRPM6 and TRPM7 association with Ca +, Mg +, Mg ATP, annexin I and myosins could be involved in regulating membrane bound cortical actin structure. Most ofmyosins' physiological functions are performed in association with actin filaments in a
2 2 manner dependent upon, and regulated by, Ca +, Mg + and Mg ATP. Each actin molecule
2 contains Mg + with either ATP or ADP (for review see Lodish et aI., 2000). The actin cytoskeleton directly effects cell morphology, adhesion, aggregation and cell cycle. Thus, a TRPM7 over-expression induced dysfunction of the actin cytoskeleton could be involved in TRPM7 over-expression effects.
Experiments were conducted to test the hypothesis that TRPM7 over-expression- induced changes in cell morphology and adhesion are the result of I) channel over-
2 2 expression causing an over-loading of the cell with Mg + or Ca + and/or 2) kinase over- expression and/or 3) possible interactions of TRPM7 over-expression effects with annexin I or the actin cytoskeleton. The channel divalent cation conductance influence on
TRPM7 over-expressing cells morphology and adhesion was observed by incubating
2 2 2 2 cells in concentrations of high or low Ca + or high Mg + or low Ca + and Mg +. The involvement of the a-kinase in TRPM7 over-expression effects was examined in cells over-expressing mutants of TRPM7 with the kinase domain deleted or made non- functional, but structurally intact. Over-expression of annexin I or mutated annexin I to be continually active or inactive Annexin I was examined in TRPM7 over-expressing cells. A link between the cortical actin cytoskeleton and TRPM7 channel was studied electrophysiologically on over-expressing cells pharmacologically manipulated with an actin toxin, cytochalasin D. TRPM7 over-expression effects on cell vitality was observed 14 with trypan-blue stain, and effect on cell adhesion was characterized by treating the cover-slips with poly-L-Iysine.
104. TRPM6 and TRPM7 OSMOSENSITIVITY
The control of water flux in and out of a cell and subsequent volume changes is crucial to cell survival. Cells regulate volume and water flux by regulating water permeability and intracellular osmolarity. Water travels through membrane hydrophilic pores down its osmotic gradient until the osmolarities are relatively equal on both sides of the membrane (isotonic). When intracellular osmolarity is higher than extracellular
(hypotonic), water floods into the cell causing swelling and when intracellular osmolarity is less than extracellular (hypertonic), water rushes out ofthe cell and the cell shrinks (for a review of osmolarity biophysics and cell physiology see Baumgarten & Feher, 2001).
Extracellular osmolarity is tightly regulated and maintained in mammals (285-310 mOsm). Most cells are water permeable and cellular osmolarity is isotonic. However, osmotic differences do occur between the inside and outside of the cell, from normal metabolic and functional activities of the cell, as well as pathological events, such as shock, ischemia, diabetes, etc. Cells must adapt to these osmotic stressors to maintain stable cell volume and not to disrupt the cell membrane, the cytoskeleton and other structures. Hypertonic cell shrinking is counteracted by an inward flux ofsmall ions (Na+ and Cn and small organic molecules. The concentrated cytosol reverses the osmotic gradient and water flows into the cell and volume is re-established. Conversely, hypotonic cell volume is reestablished by diluting the cytosol with an efflux ofMg ATP, small organic molecules and the opening of specific membrane channels to allow efflux 15 of small ions (K+ and Cr). The diluted cytosol reverses the osmotic gradient and water flows out ofthe cell and volume is re-established.
The cellular and molecular mechanisms allowing cells to sense, and respond to osmotic stressors are not completely understood. Most physical stimuli are sensed by membrane bound protein receptors, which undergo conformational changes when stimulated" These include ligand-gated, voltage-gated, stretch-sensitive, photo- and putative temperature-sensitive receptors. Certain osmoreceptors may detect stimuli associated directly with the osmotic pressure, osmotic gradient or extracellular osmolyte concentrations, such as ionic strength, hydration of protein and lipid membrane components, salinity and pressure. Other osmoreceptors could be detecting secondary stimuli resulting from intracellular changes in water activity and cell volume, such as turgor pressure, membrane and cytoskeletal strain. Stretch, ionic strength and osmotic changes in pH have been suggested to be stimuli involved in hyposmotic-induced signaling and osmolyte efflux (for reviews of bacteria, yeast and vertebrate osmosensors see Wood, 1999; Hohmann, 2002; Baumgarten & Feher, 2001; Okada et aI., 2001; Patel
& Honore, 2001).
Kinases and ion channels coupled directly to the receptor, or to a receptor activated second messenger, are implicated in osmotic stress signaling. Yeast signaling ofosmotic stressors involves osmosensitive, membrane bound kinases to initiate the weIl- characterized yeast high osmotic glycerol (HOG) MAP kinase signal transduction cascade. Similarly, vertebrate hyperosmotic stress initiates ERK5, p38 and JNK MAP kinase signal transduction cascades to phosphoregulate transcription factors, such as
OREBP/TonEBP (Ko et aI., 2002; for review see Sheikh-Hamad & Gustin, 2004). 16 TRPM6 and TRPM7 contain intrinsic cytosolic kinases and could be the mammalian initiator of the hypertonic stress signaling cascades (Nadler, et aI., 2001; Runnels et aI.,
2001; Yamaguchi et aI., 2001).
2 2 Often, cells increase [Ca +]i to signal hypotonic induced cell swelling. In yeast, a Ca +
2 channel is believed to mediate the hypotonic induced [Ca +]i increase (Batiza et aI., 1996;
Paidhungat & Garrett, 1997; Kanzaki et aI., 1999; Hohmann, 2002). Certain C. elegans
2 TRP channels sense osmolarity with altered Ca + conductance (Colbert et aI., 1997).
2 Mammalian TRP channels, TRPV4 and TRPM3, increase Ca + conductance in response to hypotonic stress, while another TRP channel, TRPC1, responds to direct
2 mechanosensation with increased Ca + conductance (Strotmann et aI., 2000; Liedtke et aI., 2000; Grimm et aI., 2003; Maroto et aI., 2005). TRPV4 is predominantly expressed in the brain and the kidneys and has been implicated as a molecular component for mammalian circumventricular organs of the hypothalamus to sense plasma osmolarity
(Strotmann et aI., 2000; Liedtke et aI., 2000; Bourque & Oliet, 1997). TRPM3 ion conductance is regulated by hypotonic stress and sphingolipid metabolites (Grimm et aI.,
2003; Grimm et aI., 2005). However, TRPV4 and TRPM3 proteins are expressed in only a few specialized vertebrate cells (primarily in the kidneys and in the brain), thus neither can explain the relatively ubiquitous nature of cell reactions to osmotic stressors (Grimm et aI., 2003; Strotmann et aI., 2000).
TRPM7 is ubiquitously expressed in all characterized mammal tissues, shares a high homology of aligned amino acid sequence to the TRPM3 sequence suggested by Grimm et aI., (2003) and has an N-terminal tyrosine kinase phosphorylation site in a similar location to the site involved in TRPV4 osmosensitive regulation (Nadler et aI., 2001; Xu 17 et aI., 2003). Conceivably, TRPM7 is involved in sensing and signaling cellular osmotic stressors or the osmolarity associated changes in cell volume and tension between the cytoskeleton and the plasma membrane.
TRPM7 a-kinase phosphoregulates annexin I and might phosphoregulate myosins.
These proteins are involved in cell structure and adaptation to osmotic stressors, such as the cytoskeleton and plasma membrane tension adjustments (for review see Gerke &
Moss, 2001 and Lodish et aI., 2000). TRPM7 is suggested to regulate cell cycle maybe by regulating cell volume, intracellular Mg2+, cytoskeleton and plasma membrane tension adjustments in the dividing or differentiating cell.
TRPM6 is involved in intestinal and renal Mg2+ (re) absorption (Voets et aI., 2004;
Chubanov et aI., 2004). TRPM6 expressing cells in the intestine and the renal distal convoluted tubule (DCT) can be exposed to extreme osmotic and ionic concentrations in the luminal fluid. The thick ascending limb of the loop of Henle and DCT transport of
Na+, K+ and cr out of the lumen fluid, causing DCT lumen fluid to become relatively devoid ofNa+ and hypotonic, less than 100 mOsm (Ganong, 2001).
The hypothesis of TRPM7 a-kinase and channel Ca2+ and Mg2+ conductance being involved in the mammalian cellular sensing and signaling osmotic stressors and or associated cell volume changes, membrane strain and mechanosensation was examined electrophysiologically. The hypothesis that, TRPM6 and TRPM617 could conduct Mg2+ at different extracellular Na+ and osmolar concentrations was also tested electrophysiologically. Whole-cell patch clamp analysis was performed on cells over- expressing recombinant TRPM7, TRPM6, both TRPM6 and TRPM7, and a kinase deletion mutant of TRPM7 with application of hyper-osmotic or hypo-osmotic 18 extracellular or intracellular fluid. In certain experiments, volume was adjusted during isotonic or hypertonic extracellular applications by balloon patch. The possibility of
2 TRPM7 involvement in cell volume re-establishment and Ca + signaling to osmotic stress
2 was assessed with the volume and Ca + indicator fluorescent dye, fura-2 AM.
1.5. SYNOPSIS OF OBJECTIVES
2 2 The proteins TRPM6 and TRPM7 form channels, that specifically carry Mg +, Ca + and other divalent cations into the cell. In addition they form a-kinase dimmers, which that auto-phosphorylates TRPM7, phosphorylates annexin I and possibly other proteins
(Nadler et aI., 2001; Runnels et aI, 2001; Yamaguchi et aI., 2001; Monteilh-Zoller et aI.,
2003; Dorovkov et aI., 2004; Ryazanova et aI, 2004; Schmitz et aI., 2004; Voets et aI.,
2 2004). In order to further understand TRPMTs ability to transport Mg +, the partial divalent cation permeation block at small positive potentials for TRPM7 was investigated using the zwitterion glutamate. Cells over-expressing TRPM7 become round, swell, clump and detach (Nadler et aI., 2001). The involvement ofthe channel and the kinase in inducing these effects was examined using various recombinant expression systems.
Osmotic stress signaling may involve plasma membrane kinases and channels, other TRP channels are osmosensitive and TRPM6 is expressed in cells likely experiencing steep transcellular osmotic gradients (Strotmann et aI., 2000; Liedtke et aI., 2000; Okada et aI.,
2001; Patel & Honore, 2001; Grimm et aI., 2003; Sheikh-Hamad & Gustin, 2004; Voets et aI., 2004; Maroto et aI., 2005). Therefore, TRPM6 and TRPM7 osmosensitivity was explored. 19 CHAPTER 2. MOLECULAR CHARACTERISTICS
2.1. TRANSIENT RECEPTOR POTENTIAL (TRP) CHANNELS
2.1.1. Transient Receptor Potential (TRP) Channels
TRPM6 and TRPM7 proteins are members of the TRP superfamily determined by sequence homology to a Drosophila retina channel involved in the sustained (T)ransient photo-(R)eceptor (P)otentials originally published by Cosens and Manning (1969) and cloned by Montell and Rubin (1989) (Referenced In Huang, 2004). TRP proteins are
2 2 cation channels that conduct Ca + and or are regulated by Ca +. TRP channels are predicted to form tetramers and contain six transmembrane domains (TM) with an ion filter and pore region between the fifth and sixth TM, similar to the Streptomyces K+ channel (Doyle et aI., 1998). TRP superfamily is primarily defined by TM similarity, instead ofthe common nomenclature grouping by channel function. The amino acids that are not part of the TM pore regions are cytosolic and define the TRP family subclasses.
TRP and TRP-1ike channels are found in vertebrates, invertebrates, plants, protozoa, fungi and even bacteria. In figure 2.1, the sequence similarity of TRP and TRP-like proteins in human, C. elegans, yeast, bacteria, Leishmania and the canonic Drosophila are compared (sequences from NCBI database).
Mammalian TRP have three subfamilies based on sequence homology, TRPC, TRPV and TRPM (for review see Montell et aI., 2002). Additional subfamilies, TRPP, TRPML and TRPA, have been suggested to accommodate novel unique TRP channel discoveries.
Figure 2.2 displays human TRP channels (and rat TRPC2) sequence similarities. 20 TRPC channels are named canonical by their similarity to the originally characterized
Drosophila TRP channel. They contain three to four ankyrin repeats in the N-terminal
2 span, 25 conserved amino acids called the "TRP box", and conduct Ca + and other cations by phospholipase C activation. TRPV channels are vanilloid by TRPVl binding to the chile pepper vannilyl alkaloid, capsaicin. TRPV contain three to four N-terminal
2 ankyrin repeats, the "TRP box" and conduct Ca +. TRPVl, 2, 3 and 4 are
2 thermosensitive. TRPV5 and TRPV6 (re-) absorb Ca + in the kidney and intestine. TRPP channels or polycystins are named for TRPP2 mutations linked to a polycystic kidney disease. TRPP contain an EF-hand motif and a C-terminal coiled-coil region, but have no ankyrin repeats or "TRP box". TRPPI is not really a TRP channel, because it has 11 TM, but the last six are similar to other TRPP channels. TRPPI and 2 are suggested to be
2 involved in cilia mechanosensation and Ca + influx. TRPML channels or mucolipins are named for TRPMLI linked to mucolipidosis type IV, an eye lysosomal storage disease and may function in intracellular vesicle trafficking. TRPML3 mutations are linked with deafness and loss ofbalance, suggesting a role in hair cell cilium transduction. TRPAI or
ANKTMI (ankyrin-!ransmembrane protein 1) has 15 to 16 ankyrin repeats on the N- terminalis span and is sensitive to cold, icillin, mustard oils and cannabinoids (for review see Huang, 2004). 21
ClustalW (vl.4) Multiple Alignment Parameters: Open Gap Penalty - 10.0; Extend Gap Penalty ~ 0.0; Delay Divergent ~ 40% Gap Distance"", 8; Similarity Matrix = id
O.,:!'1TRPC3 TRPC hTRPC6 ceTRP1 ceTRP2 dmTRP hTRPC5
O.2d2TRPC4 hTRPC1
rTRPC2 ceGTL2
O.3&GON2 ceGTL1 TRPM
hTRPM1
hTRPM4 '------' scTRPY1
mgPTRP
leshTRPN1
TRPP I
TRPAIN I
ceOsm9 ceOCR4 ceOcr3 ceOcr2 ceoeR1
::::::hTRPV4 I mv I hTRPV2 hTRPV1 hTRPV3 0.402 ceCED11
ecZ65
0.17 hTRPML~ O.2./l,TRPML TRPML hTRPML I
Figure 2.1 A guide tree representing human (h) and C. elegans (ce) TRP protein amino acid sequence similarity, as well as, rat TRPC2, the original Drosophila TRP and TRP like proteins of yeast (sc, mg), e. coli (ec) and Leishmania (lesh). Identity scores are based on ClustalW pairwise and multiple pair array analysis of gapped amino acid sequences alignments. 22
ClustalW (vl.4) Multiple Alignment Parameters: Open Gap Penalty = 10.0; Extend Gap Penalty = 0.0; Delay Divergent = 40% Gap Distance = 8; Similarity Matrix = id
hTRPV6 hTRPVS hTRPV4 hTRPV2
hTRPV1 hTRPV3
hTRPA1 hTRPPS hTRPP3 hTRPP2a hTRPC7
hTRPC3 hTRPC6 hTRPCS hTRPC4 hTRPC1 rTRPC2 hTRPML3 hTRPML2
hTRPML1 hTRPM8 hTRPM2
hTRPMS hTRPM4 hTRPM7 hTRPM6 hTRPM3 hTRPM1
Figure 2.2 A guide tree representing human TRP protein amino acid sequence similarity (and rat TRPC2). The identity scores are based on ClustalW pairwise and multiple pair array analysis ofgapped amino acid sequences alignments. 23 2.1.2. Melastatin-like Transient Receptor Channels (TRPM)
TRPM6 and TRPM7 are members of the TRPM subfamily. TRPM channels are very homologous, especially from the N-terminal to about 200 amino acids on the C-terminal side ofthe sixth TM (see Appendix 1 and Table 2.1). The amino acid sequence alignment tree in Figure 2.3 illustrates, that the TRPM subfamily forms two subgroups, one group contains TRPM1, TRPM3, TRPM6 and TRPM7 and the other group contains TRPM2,
TRPM4, TRPM5 and TRPM8.
TRPM are named for TRPM1 or melastatin. Its expression exhibits an inverse correlation with melanoma aggressiveness (Duncan et aI., 1998). TRPMl may conduct
2 Ca +, but the physiology and electrophysiology ofthe channel is still unknown (Xu et aI.,
2001; Duncan et aI., 1998). At least nine related genetic sequences claim to be TRPM3 in the NCBI database. One ofthese is activated with the sphingolipid metabolite, D-erythro
2 sphingosine, to conduct cations with a preference for Ca + and is correlated with increased [Ca2+]i in response to hypotonic solutions (Grimm et aI., 2003; 2004). The similarity of TRPMl and TRPM3 with TRPM6 and TRPM7, especially in regions speculated to be involved in tetramer formation, suggests that these channels may form heteromers.
Two variants of TRPM4 have been discovered, TRPM4a and TRPM4b. TRPM4a is
2 associated with a Ca + influx (Xu et aI., 2001, Murakami et aI., 2003). TRPM4b and
2 TRPM5 are monovalent cation-specific channels that do not conduct Ca +, but whose activities are regulated by [Ca2+]i (Launay et aI., 2002; Prawitt et aI., 2003; Liu & Liman,
2003; Hofmann et aI., 2003). Prolonged, slow [Ca2+]i increases activate TRPM4b, while
TRPM5 activation is due to a fast transient bolus of [Ca2+]i, followed by rapid 24 inactivation, suggesting involvement ofTRPM5 in fast signaling and TRPM4b in gradual increases in [Ca2+Ji. TRPM4b is ubiquitous and regulates Ca2+oscillations in activated T- lymphocytes (Launay et al., 2004). TRPM5 is found in the digestive system and is implicated in bitter sensation (Perez et al., 2002). TRPM4 and TRPM5 are suggested to form heteroligomers (Earley et a1., 2004).
TRPM2 contains a nudix hydrolase domain and the channel is gated by the NAD metabolite ADPR, which also interacts with the nudix enzyme (Perraud et al., 2001; Shen et al., 2003). TRPM2 is expressed in the nervous, digestive and immune system and its non-specific current may function in oxidative stress and apoptosis signaling (Hara et a1.,
2002; Wehage et al., 2002; Nagamine et al., 1998). TRPM2 chromosomal locus is associated with bipolar disorder and a subset of bipolar disorder patients were shown to have low TRPM2 mRNA translated in their B-lymphocytes (Aita et al., 1999; Yoon et al., 2001). TRPM8 is likely involved with the sensation of cold and the "cool" of menthol, while prostate TRPM8 may have a role in cancer (Peier et a1., 2002; Zhang &
Barritt, 2004) (for review see Fleig & Penner, 2004)
The key protein structure separating TRPM6 and TRPM7 from all other channels is the cytosolic carboxyl terminal a-kinase. a-kinases exist in protozoa, worms, fungi as well as mammals. Surprisingly, a-kinases have not been found in the genomes of yeast, insects or higher plants. In humans six a-kinases have been discovered, elongation factor-2 kinase, TRPM6, TRPM7, a lymphocyte a-kinase and two a-kinases believed specific to cardiac and skeletal muscles (for review see Ryazanov, 2002 and Drennan &
Ryazanov, 2004). 25 Table 2.1 The percentage of each of the human TRPM protein sequences that align identically or similarly with TRPM3 sequence (Grimm, 2003). Sequence alignments were adjusted with gaps.
Sequence TRPMl TRPM2 TRPM4 TRPM5 TRPM6 TRPM7 TRPM8
Identical 57% 24% 25% 23% 33% 39% 22%
Similar 9% 16% 17% 17% 11% 12% 17%
ClustalW (vl.4) Multiple Alignment Parameters: Open Gap Penalty = 10.0; Extend Gap Penalty = 0.0; Delay Divergent = 40% Gap Distance = 8; Similarity Matrix = id
hTRPM5 hTRPM4 0.279 hTRPM2 hTRPM8 hTRPM7 hTRPM6 hTRPM3 hTRPM1
Figure 2.3 A guide tree representing human TRPM proteins amino acid sequence similarity. The identity scores are based on ClustalW pairwise and multiple pair array analysis ofgapped amino acid sequences alignments.
2.2. TRPM6 and TRPM7 PRIMARY STRUCTURE
2.2.1. PROSCAN Putative Functional Motifs
All the characteristics ofproteins are determined by their amino acid sequence, their modifications and interactions. There has not been found any functional differences between TRPM6 and TRPM7, other than heredity studies and tissue distribution of proteins and mRNA. The two proteins have remarkably similar amino acid sequences 26 with each other and all TRPM proteins, especially the amino-terminal span (N-t span), the putative transmembrane (TM) domains, the cation pore, the carboxyl-terminal
"TRPM box" region and the a-kinase (see Appendix 2.1 for the amino acid alignments between TRPM proteins). However, specific differences do exist and may provide clues to the functions and regulations ofthese channels.
Certain amino acid sequences or patterns are identified to be conserved through-out biology in a variety of different proteins, mainly for their ability to perform specific functions. Specific TRPM6 and TRPM7 amino acid sequences were found to be 80% similar to the amino acid sequences defining such motifs in the Prosite motif database by an online search program, PROSCAN, http://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.pl? page=/NPSA/npsa server.html (Bucher & Bairoch, 1994).
These motifs found suggest interactions of these proteins with divalent cations, regulation by kinases and interactions with the plasma membrane. None of these sequences in either TRPM6 or TRPM7 have been proven to have any function. Many motifs align at similar region of TRPM6 and TRPM7, especially glycosylation and myristoylation sites. However, many motifs are different between the two proteins and may provide further insight into the purpose of having such similar proteins expressed in the body and how they differ physiologically (see table 2.2). These differences in motifs are usually by one or two amino acids crucial to the motif function in the aligned sequences. In appendix II the amino acid sequence alignment of TRPM6 and TRPM7 is illustrated with motifs indicated and in appendix III a brief description of the motifs alleged functional profiles is provided. 27 Table 2.2 Functional motifs determined with PROSCAN to be more than 80% identical to sequences in TRPM6 (M6) or TRPM7 (M7). TM indicates the motif is in the transmembrane region, N-t for N-terminal span and C-t for C-terminal span of the respectIve..protem. The motlof:s are explI'amed'10 append'IX 23.. Motif InM6 InM7 Re/!ion ofProtein TyrKinase Phospho-site 0 3 coiled-coil, kinase, mid N-t cA/GMP Kinase Phospho-site 5 42 N-t, 2 Non-similar C-t, coil-coiled (M6) Protein KinaseC Phospho-site 29 25 Casein Kinase II Phospho-site 41 30 Heme-bindinj!, peroxidase 1 ON-t EF-hand Ca-binding 0 1Just N-t ofTMI EGF-like domain sig 1 0 1Beginning ofN-t ZnCarboxylase Zn-bind 2 sig 1 ON-t ATP-bind Protein kinase sig 1 1Non-similar C-t (M6),Beginning N-t (M?) AMP-bind site DNA ligase 0 1C-t coiled-coil TyrK P site N-glycosylation 15 12 N-myristoylation 27 30 G-protein recept signature 4 I N6A-DNA Methylase sig 1 oTM3 to TM4 excell loop (M6) Trp-Asp (WD) repeats sig 1 1N-t (M6); TM2 to TM3 loop (M7) C-type lectin sig 0 1Beginning ofN-t Cys proteases inhibitors 1 oBegin ofkinase PyrrolidoneC03 ase glu 0 1Kinase Heavy-metaV Kringle I 1Kinase, Zn chelation sites Hemopexin 0 1N-t Pancreatic ribonuclease sig 1 ON-t Unknown stressor 0 I Just prior to TMI on N-t side C-Phospho synthase sig 2 I oC-t coiled-coil (M6) Phosphopantetheine 1 I N-t (M7), Non-similar C-t (M6) Glycosyl hydrolases 1 0 I N-t Glycosyl hydrolases 11 sig2 0 I Non-similar C-t (M7) 28 2.2.2. Amino-terminal Region ofTRPM6 and TRPM7
TRPM6 and TRPM7 are remarkably similar in the amino-terminal span (N-t span), the putative transmembrane (TM) domains, the cation channel domain, the carboxyl-terminal
"TRPM box" region and the a-kinase. Figure 2.4 illustrates the N-t span amino acid sequence alignment of TRPM6 and TRPM7. Note the aligned coiled-coil region and the
EF-hand like region at the end of TRPM7 N-t span, suggesting a possible influence of
2 Ca + on TRPM7, but not TRPM6. The serine involved in putative tetramer formation and mutated in a TRPM6 associated hypomagnesemia is colored. TRPM7 tyrosine kinase phosphorylation (TKP) site is in a similar location as a TRPV4 TKP site regulating osmosensitivity, although the surrounding amino acids are different (Xu et aI., 2003).
TRPM6 and TRPM7 osmosensitivity is examined in this dissertation. M6 fqfmwyysdqnassskesasvkeydlerghdekldenqhfglesghqh M7 ahqmtmOdsennfqnt~eeipmevfkevrildsnegknemeiqmkskk Figure 2.4. TRPM6 and TRPM7 amino acid sequence alignment for the amino-terminal span. Amino acids are color-coordinated for residue chemistry. The highlighted amino acids are, a serine speculated to be involved in heteromerization (blue), a tyrosine kinase phosphorylation site (red), cAMP/cGMP protein kinase phosphorylation sites (yellow) and coiled-coil regions associated with EF-hand Ca2+-binding sites (grey).
2.2.3. Putative Transmembrane Region ofTRPM6 and TRPM7
The putative TM domains were determined for the sequences of TRPM6 and TRPM7 by an educated adaptation of hydrophobicity, surface probability and transmembrane 30 probabilities from various mathematical analyses of amino acid residue chemistry performed by MacVector software; Argos and von Heijne transmembrane probabilities,
Sweet/Eisenberg, Fauchere/Pliska and Janin hydrophobicity scores, and surface probability. These tests model a general probability of the amino acids forming a TM domain, but they do not provide exact domains. TM domains are thought to exist in a- helical secondary structures. The primary sequences of the proposed domains are illustrated in figure 2.6. They are not expressed as interlocking helical wheels, because the actual sequences and interacting amino acids ofthese helices is not clear.
TRPM7 contains a umque sequence of aromatic amInO acids,
FGAKWFNAFANAYDNHV in humans and FGAKWNYINAYDNHV in the mouse, which is located either as an extracellular loop between the 3rd and 4th TM or as an extension of one ofthese domains. Unique extracellular sequences have the possibility to interact with the environment, such as other cells, binding ligands or being specifically targeted by drugs or antibodies. The 4th transmembrane domain of distant relatives of
TRP channels, the voltage-gated channels, is crucial for opening the channel in response to changes in voltage potentia1. Possibly, the above sequence is an elongation ofTRPM7
4th TM domain and could be a method for sensing stimuli, such as pressure. TRPM7 ability to sense osmotic pressure is examined in later chapters.
TRP proteins form channels selectively conducting cations, usually including Ca2+.
The predicted selectivity filter and pore region ofTRP channels are believed to be similar to the crystallized Streptomyces lividans K+ channel (Figure 2.5). Similar to certain K+ channels, this region is likely formed by the amino acids between and including the 5th and 6th TM of each protein in a putative tetramer. Cation selective filters in other 31 channels are based on mimicry and stripping of the cation hydration shell by glycine peptidyl-amide oxygens stabilized with an aromatic residue, such as tyrosine or phenylalanine (for review of ion pore theory see Hille, 2001). Two glycines are conserved in all TRPM pore regions, e.g. in TRPM6 these are in the IsfGv and the miyGev sequences.
Figure 2.5. Tube representations of Streptomyces K+ channel pore region and transmembrane domains surmised by X-ray crystallography (Zhou et aI., 2001).
All TRPM channels share a similar amino acid sequence in the filter-pore region.
However, the cations and the ratio ofthe different cations conducted differ for each ofthe characterized TRPM channels. TRPM6 and TRPM7 are specific for divalent cations at negative potentials, TRPM2, TRPM8 and TRPM3 carry non-selective cation currents,
2 while TRPM4 and TRPM5 do not conduct Ca + or other divalent cations. TRPM1 channel conductance has never been characterized (for review see Fleig & Penner,
2004). There are similar unique amino acid sequences in the filter/pore region shared by channels ofsimilar conductance that differ from the other channels. These differences are 32 unclear and sporadic (Figure 2.7). Surprisingly, the region corresponding perfectly with channel conductance is the putative 6th TM domain (Figure 2.8).
The sixth TM amino acid sequence separates into identical or nearly identical pairs ofTRPM6 & TRPM7, TRPMI & TRPM3, TRPM4 & TRPM5, and TRPM2 & TRPM8.
The pairing corresponds perfectly with the electrophysiological conductance characteristics of these channels. However, without either X-ray crystallography or site- directed mutagenesis, this information is only anecdotal.
~? M7? ~~ M7~ t-f5? M7? ~~ M7~ ~? M7? ? 1 f ? 1 f ~~ M7~
~ g111rrl-cchraphdqeegdvglklylskedlkklh M7 1f . k kkdkt dIIkl teecJqkklh t-f5 dfeeqevekyfhekmedvncsceeri. t 5 5 5 S M7 dfeeqev~fnekddkfhsgseeri. • SSS M5 lsaltvdtlkvlsa M7 lsaltvdtlktlta Figure 2.6. Transmebrane domains (boxes), ion pore and conserved C-terminal span to coiled-coil region (green). Amino acids are color-coordinated for residue group chemistry. The highlighted amino acids are unique extracellular loop or transmembrane elongation of TRPM7 (grey), aromatic-glycine possible cation filter sites (yellow), the "TRPM box" (teal), the coiled-coil region and a tyrosine kinase phosphorylation site (red). The arrows indicate the peptide sequence direction spanning the membrane. 33 TRPM6 lsfgvarkailspkeppswslardivfepywmiygevyage-id TRPM7 1s~ prkai.lyph ps lakdivfhpyYlfl11 fge aye-id TRPM3 ~ arqai.lfpneepswk1akni. fyrrp .ygevfa-dqid TRPMl msfgvarqailhpeekpswklarnifyrrpyYlfl1i.ygevfa-dqi.d TRPMZ vs~ akqai.1ihnerrvdw1frgavyhsy1~lfgqlpgy--id TRPM8 va~ arqgllrqneqrw .frsviyepyl gqvp d-vd TRPM4 vaygvategllrprdsdfpsilrrvfyrpylqifgqipq-edmd TRPMS va~ tqallhphdgrlewifrrvlyrpylqifgqip1-deid
Figure 2.7. Putative amino acid alignments ofTRPM channels cation selectivity filter and pore region. Amino acids are color coordinated by residue chemistry. Aromatic-glycine residues believed to be crucial for cation pores are highlighted.
qyi.imvn11laffnn 1 qyi.lmvn11iaffnn 1
TR PM3 goo .rracy11vanil1vn11i fnntff TRPMl gawltpalmacyllvanillvnlliavfnntff
TRPMZ pew1 vlllclyllf nl1llnlli.anfn fq TRPM8 .tiplvciym1stni11vn1lvanfgyt"vg
TRPM4 anw1vvl1lvifl1vanillvnl1iamfs fg TRPMS anw1vi.l11 1vtnv11mnlli.amfs fq
Figure 2.8. The putative sixth transmembrane domain ofTRPM channels and a portion of an extracellular loop alignment of amino acids. Amino acids are color coordinated by residue chemistry.
2.2.4. Carboxyl-terminal Region ofTRPM6 and TRPM7
Directly on the C-terminal side of the 6th TM is the "TRPM box". This conserved amino acid sequence shares similarity in location and identity with the "TRP box" of
TRPV and TRPC proteins. In TRPMl, 3, 6 and 7 an aligned coiled-coil region is predicted to follow the "TRPM box" http://npsa-pbil.ibcp.fr/cgi- 34 binlnpsa automat.pl?page=/NPSA/npsa lupas.htm (Lupas et al., 1991). Within TRPM7 coiled-coil region is a tyrosine kinase phosphorylation site.
Following the coiled-coil region until the carboxyl-terminal a-kinase, there is a sequence of 473 amino acids in TRPM6 and 294 in TRPM7, completely different from each other and other TRP proteins. The residues are primarily serines, threonines and prolines. TRPM6 sequences are not conserved between mice and humans, suggesting the spans could be just an aqueous separation of the kinase from the membrane and the channel region (56% identity, 67% positives & 9% gaps). TRPM7 sequences are more conserved between human and mouse (79% identity, 86% positives & 0 gaps). Perhaps, the TRPM7 span performs a function other than separation. TRPM7 a-kinase in vitro auto-phosphorylation is greatly enhanced by including this serine/threonine rich region, suggesting this region is auto-phosphorylated (Yamaguchi et al., 2001). Both TRPM6 and
TRPM7 spans predict a coiled-coil region and cAMP/cOMP-kinase phosphorylation sites
(figure 2.11).
The carboxyl-terminal a-kinases are similar to conventional protein kinases (CPK) in tertiary protein folding and in the ability to phosphorylate serine and threonine hydroxyIs using phosphate derived from ATP, but differ from CPK in primary amino acid sequence and phosphorylation of these residues within a-helical secondary structures. a-kinases are conserved within and between species with approximately 35% identity and 60% similarity ofaligned amino acids. As shown in figure 2.10, TRPM6 and TRPM7 a-kinase domains are essentially identical. (Yamaguchi et al., 2001; for review see Drennan &
Ryazanov, 2004). 35 Yamaguchi et al. (2001) obtained an X-ray crystal secondary structure ofTRPM7 a- kinase (see figure 2.9). The ATP binding cleft is more comparable to metabolic enzymes
ATP grasping sites than CPK ATP binding sites. The catalytic transfer of a phosphate from ATP to the target hydroxyl of serine/threonine for a-kinases is believed to be
2 similar to CPK but the interactions ofkey charged residues with Mg + and the phosphates are not entirely clear. The glycine rich loop appears to be flexible and is in a location suggesting substrate recognition. A conserved C-terminal zinc-finger is probably important for structure.
Figure 2.9. TRPM7 a-kinase domain secondary structure from X-ray crystallography by Yamaguchi et al. (200 I). The glycine rich loop is indicated, the ball and stick molecule in 2 the middle is ATP in the ATP grasping fold, the Zn + ions are in structures like Zn fingers, a-helices are represented as rod shaped arrows and b-sheets are tape shaped 2 arrows. The Zn + and ATP stick-&-ball model on the left are to appreciate the dimer structure ofthe kinase. 36
fJ6 dl M7 1 fJ6 dlkrndysperi. 1 . fJ6 nstfglei.ki.esaeepparet-grnspeddmql ln1 v 1- -1
Figure 2.10. TRPM6 and TRPM7 a-kinase and C-terminal amino acid sequence alignment. Highlighted amino acids are the beginning of a kinase deletion mutation (blue), single-point mutations (pink), tyrosine kinase phosphorylation site (red), the phosphotransfer site (yellow) and a crucial glycine-rich loop (box).
Figure 2.11 Unique TRPM6 and TRPM7 amino acid sequence span. Highlighted amino acids are threonines and serines (black), proline (red), cA/GMP kinase phosphorylation sites (yellow followed by threonine or serine), and a coiled-coil region (grey). 37 CHAPTER 3. MATERIALS AND METHODS
3.1. CELLS
TRPM7 channel expression is ubiquitous (Nadler, 2001). The endogenous TRPM7 was examined in the HEK-293 cell-line. HEK-293 is a fibroblast-like epithelial cell-line originally derived from primary human embryonic kidney (HEK) cells transformed with
DNA sheared from type 5 adenovirus (Shaw et aI., 2002). Transformation allows the cells to be continuously cultured for a number of passages. HEK-293 cells were cultured in appropriate media (see section3.2) and passed three times a week. Cells were grown on glass cover-slips untreated or pre-treated with 0.1 mg/ml poly-L-Iysine hydrobromide
(Sigma, MO) (Card, 2000).
In certain experiments, HEK-293 chromosomes were stably incorporated with genes encoding a tetracycline responsive gene expression system coupled with one of the following genes: mouse TRPM7 from the Jean-Pierre Kinet laboratory in Boston, MA; human TRPM7 or mutated human TRPM7 with the kinase not expressed or single-point mutated human TRPM7 of a glycine (1599) to a glutamate or a lysine to an arginine
(1648) from the Andrew Scharenberg laboratory in Seattle, WA; human TRPM6 or both
TRPM6 and human TRPM7 or both TRPM6 and mutated kinase-deleted TRPM7 from
Anne-Laure Perraud's laboratory in Denver, CO respectively; and human TRPM7 with
GFP motif, both human TRPM7-GFP with human annexin 1 or both human TRPM7 and mutated annexin 1 of a serine (5) to an alanine, or both human TRPM7 and mutated 38 annexm I of a senne (5) to a aspartate from the Alexey Ryazanov laboratory in
Piscataway, NJ (Nadler et aI., 2001; Schmitz et aI., 2004; Dorovkov et aI., 2004).
Expression ofthe target gene(s) was induced for 14-72 hours with 1 !-Ig/ml tetracycline added to the medium to initiate the tetracycline responsive gene expression system. This system utilizies the tetracycline-operator (TetO) derived from the TN 1O-tetracycline resistance operon of E. coli. TetO-mediated gene transcription is repressed by the tetracycline repressor protein (TetR). TetR gene is always expressed by being fused with a human cytomegalovirus immediate early gene 1 enhancer/promoter. Tetracycline unbinds TetR from TetO and this allows TetO-mediated gene transcription to occur, in this case, the over expression of the target channel gene (Gossen & Bujard, 1992).
Success of the target expression in the plasma membrane was examined electrophysiologically and by immunocytochemical staining for a sequence of amino acids encoded to the transfected gene and expressed in the protein with readily available anti-bodies (data not shown).
Human TRPM6 eDNA coupled to a green fluorescent protein motif tag (GFP) from the Bernd Nilius laboratory in Belgium was examined in HEK-293 cells using MIRUS
TransIT ®-LTI (Mirus-Bio, WI) transient transfection reagent (Voets et aI., 2003). Cells were plated at a density of400,000 cells/ml in DMEM. A mixture ofTRPM6-GFP eDNA
(2 !-Ig) incubated in TransIT ®-LTl reagent (6 !-II) and DMEM (44 !-II) was added to cells in 500 !-II DMEM and incubated for 24-48 hours. Cells were detached and individualized for electrophysiology by a pipette, pumping media up and down over the cells. The detached cells (70 !-II) were gently transfered to a Petri dish without a glass cover slip and 39 after 15-25 min, patch-clamp bath solution was added (-2ml). Cells with successful transfection and expression were identified by the fluorescence ofGFP.
3.2. SOLUTIONS
Cells were cultured in media consisting of Dulbecco's modified Eagle medium
(Gibco, NY) supplemented with 10% fetal bovine serum, penicillin/streptomycin (2.1 mL) and blasticidin (5 f!g/ml) (Invitrogen, Carlsbad, CA) in 5% CO2-humidified atmosphere at 37°C (DMEM). The DMEM for cells stably transfected with human or mouse TRPM7 was supplemented with zeocin (0.4 mg/ml) (Invitrogen, CA), while cells stably transfected with human TRPM6 were supplemented with hygromycin B (0.2 mg/ml) (Invitrogen, CA). In certain experiments, cells were incubated for 48-72 hours in
DMEM supplemented with one of the following (in mM): 10 MgCh; 9 CaCh; or appropriate amounts of sucrose to adjust osmolarity to 480 mOsm. In other experiments, cells were incubated in DMEM for 24 hours in 10 mM K2BAPTA; or 6.8 mM
Na2HEDTA with 4 mM CaCh; or 5 f!g/ml cytochalasin D (Sigma, St. Louis, MO). In 2 order to examine volume and intracellular Ca + levels, cells were incubated for 30-45 min in 5 11M Fura 2-AM (Molecular Probes, USA). Cell viability was examined by adding
0.04% trypan blue stain (Gibco, NY) to the incubation media for 45 min.
In whole-cell patch-clamp experiments, the extracellular bath solution consisted of a modified standard Ringer's solution (in mM): NaCI 140, KCI 2.8, CaCh 1, MgCh 2, glucose 10, HEPES-NaOH 10; pH 7.2, 310-330 mOsm. Hypotonic extracellular application solutions were filtered modified Ringer's solution with a low NaCI concentration (-80 mM). In these experiments, isotonic applications and bath solution 40 used the hypotonic solution and adjusted the NaCl concentration to 140 mM and the saline to 310-330 mOsm. Hypertonic application solutions consisted of filtered bath solution with osmolarity adjusted by appropriate amounts of sucrose or NaCl. Sodium glutamate application solutions contained either 140, 70 or 1 mM Na glutamate with 0,
70 and 140 mM NaCl modified Ringer's solution. Extracellular application solutions were applied by pressure ejection from wide-tipped pipettes. Divalent cation free external 2 2 solutions and application solutions were made without Ca + or Mg + and in certain experiments 5mM Na2EDTA was added. Na-glutamate and Cs-glutamate were made with glutamic acid powder brought into solution with NaOH or CsOH.
Intracellular pipette-filling solution consisted of (in mM): Cs-glutamate 140, NaCl 8,
Cs-BAPTA 10, HEPES-CsOH 10; pH 7.2, 310-330 mOsm. Hypo-osmotic intracellular pipette-filling solutions (~250 mOsm) were similar but with less Cs-glutamate (l00 mM).
Hyper-osmotic intracellular pipette-filling solutions were adjusted with additional CsCl. 2 The free Mg + concentration was adjusted with MgCh for each condition of 0, 0.4, 0.9 and 3 mM as calculated by MaxChelator software
2 MgoATP (Lot #97H7006, Sigma, MO) was added to the solution and the free Mg + concentration was appropriately adjusted. Divalent cation free internal solutions were 2 made without Mg + and in certain experiments 5mM Na2EDTA was added. Experiments 2 monitoring intracellular Ca + levels used intracellular pipette-filling solutions with 200
[!M Ks-Fura 2 (Molecular Probes, Eugene, OR) in place of BAPTA. A vapor pressure osmometer (Wescor, Utah) was used to measure the osmolality ofall the solutions. 41 3.3. PATCH-CLAMP ELECTROPHYSIOLOGY
3.3.1. Whole-cell Patch-clamp Method
The opening and closing of ion channels and subsequent currents or voltage changes can be measured for a single channel and the whole membrane of an intact cell using patch-clamp electrophysiology. The patch-clamp method uses suction to form tight seals between a glass pipette and the cell membrane and amplifiers that can allow for the measurement ofminute currents.
The recording-electrode is a saline filled, tapered, fire-polished glass pipette (Kimax
51, Kimble Products, USA) with a AgCl-coated silver-wire inside, while the bath electrode is a AgCI-coated silver-wire. The recording-electrode manipulates the voltage potential between the two electrodes by injecting or withdrawing the necessary charges.
The current signal through the electrode to produce these charges and maintain the potential is filtered at 2.3kHz and is amplified by an operational 'pre' amplifier. A voltage drop across a set resistor quantifies the current signal and a set resistor determines the gain (1-V converter). The signal is converted to a digital signal by a resistor and a computer-based amplifier system (EPC-9, HEKA, Germany) connected to a computer hard drive. Computer software (Pulse+Pulse-Fit v8.50, HEKA, Germany) records, manipulates and displays the digital signal, and allows the user to establish the experimental parameters.
A coverslip of cells is secured with silicon dabs to a 2.9 ml Petri dish and incubated in
~2 ml ofextracellular patch-clamp bath saline (see section 3.2). The dish is placed in the patch-clamp apparatus and observed by a video camera and monitor (Sony, Japan) through a microscope (Axiovert 35, Zeiss, Germany). The recording-electrode is 42 mounted and sealed in a micromanipulator apparatus-arm (Eppendorf, Germany) with access for an attached tube to apply suction or positive pressure. The recording-electrode was advanced by the micromanipulator into the bath saline and brought to gently contact a target cell. When the recording-electrode first enters into the bath saline, a voltage potential created by anions and cations having different motilities in the recording- electrode and bath solutions (liquid junction), was countered with an opposite voltage (10 mV). Voltage-clamped test pulses·are applied at +10mV and -10mV for 20 ms each and the current is recorded at a small gain (1 mVIpA). The total resistance between the electrodes is determined (~2-3 MQ). These are a series ofresistances, the most prominent being the pipette-tip size.
A slight suction coupled with the charges on the glass electrode is applied to "pull" the glass tip and cell ectoplasmic membrane together in a water-ion tight seal. The poorly conducting glass and lipid membrane form a large resistance between the electrodes of at least 1 GQ, the "gigaseal".
The small patch of membrane isolated within the hole of the pipette tip acts as a capacitor between the conductive cytosol and the pipette solution. The electrode glass also acts as a capacitor between the conductive bath and test electrode solution. In order to determine this capacitance and compensate for the currents from capacitance charging and discharging in relationship to voltage, a series of small pulses (5 mV) are applied.
The capacitance is measured by the voltage development and decay at 1Ie ofthese pulses
(-t) divided by the resistance. The resulting capacitive currents from the glass test- electrode, the membrane patch and other "stray" capacitances (C-fast) and a fudge factor for the pre-amplifier (gain = 10mVIpA) are extrapolated for the necessary current 43 (voltage) to cancel them, often by a variable capacitor (for review of patch-clamp techniques see Penner, 1993).
In the whole-cell patch-clamp mode, a quick pulse of suction breaks the membrane patch within the tip without disrupting the seal. This allows the electrode solution access to the cell, while preventing the bath solution from entering into the cell. The recording- electrode solution manipulates the intracellular contents. The clamped voltage and resulting leak current from test pulses determine the access resistance to the cell.
The non-polar cell membrane acts as a capacitor separating the charges of the conductive internal and external solutions. Similar to C-fast, the charging/discharging of the membrane at the onset and end ofa -100 to +100 mV ramp (C-slow) is compensated by the EPC-9 and or a variable capacitor by the appropriate currents being added/subtracted when initiating voltage changes.
In the experimental conditions ofthis dissertation, the voltage is clamped for 2 s at 0 mV (with the exception of test pulses examining C-slow), followed by a continuous voltage ramp from -100 to +100 mV over 50ms. The current necessary to maintain the voltage potential during the ramp is recorded every 100 !As during the voltage ramp and for 5 ms before and after the ramp at the holding potential. The certain current-voltage traces at specific pulse are analyzed and presented within the results section. The current elicited at a voltage of-80 mV and of+80 mV during the voltage ramps are analyzed and plotted. The -80 mV represents the currents to be expected during cell resting potential.
A separate software system (Igor Pro, OR) was used to compile, compare and graphically present the data. 44 3.3.2. Solution Application
Before whole-cell configuration is begun, an applicator-pipette with access for
pressure manipulation via tubing to a pressure pump (Lorenz, Germany) is filled with 20
50 !AI of hypertonic or isotonic or hypotonic or Na glutamate solution and sealed to a
manual three-axis hydraulic micromanipulator apparatus arm (Narishige, Japan). The
application pipette tip is brought in close proximity to the target cell and a portion is
broken-off by pressing it on the glass coverslip to widen its opening to ~ 10 !AM. The
pipette is removed from the bath along the vertical axis and a slight negative pressure holds the fluid in the pipette.
At about 290s after whole cell patch-clamp break-in, a positive pressure is given to the
fluid and the pipette is lowered into the bath next to the cell, thus at 300s after break-in,
the application solution flows over the cell. After 20-200 seconds, the application pipette
is removed from the bath along the vertical axis. The EPC-9 continues to record the
current for another 100-200s.
3.3.3. "Balloon" Patch Volume Application
In certain whole-cell patch-clamp experiments positive pressure is applied by mouth to
the internal fluid of the cell at ~400s after break-in. The pressure adds to the normal
hydrostatic pressure of the recording-electrode perfusion of the patched cell and is just
enough to force the recording-electrode solution to increase the cell volume and size
without exploding the cell. The pressure is removed after the increase in cell sIze
(ballooned) is achieved and the cell is allowed to deflate. 45 3.4. FURA-2: A FLUORESCENT INDICATOR OF CALCIUM AND CELL
VOLUME
In certain whole-cell patch clamp experiments, the internal solution included 200 ~M
K+ fura-2 (Molecular Probes, USA). In a different set of experiments, intact HEK-293 cells were loaded with 200 JlM fura-2 ester dissolved in DMSO (Fura-2-AM, Molecular
Probes, USA) for 60 min at 37°C in external solution containing (in mM): 101 NaCl, 6
KCI, 1 MgCh, 2 CaCh, 5 glucose and 10 HEPES-NaOH; pH 7.2; 310-330 mOsm. The esterification offura-2 cloaks the chelating carboxyl motifs, allowing for a relatively non- polar compound to cross the plasma membrane lipid bi-layer into the cytosol, where esterases hydrolyze off the acetates, forming a fully functional and polar fura-2 in the cytosol ofintact cells, which cannot escape across the plasma membrane.
Fura-2 was used to monitor intracellular Ca2+concentration changes and cell volume changes in these cells. Fura-2 is an UV-excitable fluorescent Ca2+ indicator derived from the Ca2+ chelator BAPTA. Ca2+-free fura-2 has a fluorescent excitation maximum of 363 nm, but fura-2 bound to Ca2+ shifts the fluorescent excitation maximum to 335 nm. The fluorescence emission remains relatively unchanged at -510 nm in either state. The isosbestic point, the point where there is no difference in the excitability ofCa2+-free and
Ca2+-bound fura-2 is -360 nm (See Figure 3.1).
Traditionally, fura-2 is excited at UV-light wavelengths of 360 nm and 380 nm, although sometimes 340 nm is used instead of 360 nm. The wavelength of 380 nm excites Ca2+-free fura-2 with minimal Ca2+-bound fura-2 excitation. When the [Ca2+]i changes, the emission intensity excited at 380 nm changes reflecting the ratio of Ca2+-free fura-2 and Ca2+-bound fura-2 in the cell. The isosbestic point excitation provides a 46 2 baseline of total fura-2 dye emission regardless of Ca + levels. The ratio of the emission
2 intensity at 380 nm to emission intensity at 360 nm allows for measurements of Ca + concentration in real time and minimal error due to fura-2 concentration, uneven dye distribution, photobleaching and volume changes (Grynkiewicz et aI., 1985).
43 5IJ.M free Co H
0441 0.284 o 89' 0126 0.081 '-. 0047 0021 o
250 300 350 400 450 Figure 3.1. The intensity of 512 nm light emitted by a set concentration ofFura-2 bound 2 to various Ca + concentrations in response to excitation of light wavelengths in a range from 250 nm to 450 nm. Two common analytic ponts, 340 nm light (1') or 380 nm light (-J..-) are pointed out. Notice the emission intensity is constant at 360 nm excitation (Source unknown).
There are several procedures involved in controlling the precise frequency and wavelength of the excitation light. These procedures are either performed by separate apparatus or included in a few or one apparatus. The circuit is opened and closed at a determined frequency by a switch to allow for a dark and light phase. The closed circuit- input current is altered into two different current sizes. Each current size is held for 20ms and is simultaneously received by a dual wavelength photometer and a scanner power 47 supply. The scanner power supply converts the input into a highly stabilized DC voltage.
The voltage is applied to a galvanometric scanner (T.I.L.L. Photonics, Germany), which
moves a grated mirror accordingly. Each grating on the mirror deflects a specific
wavelength oflight.
A white light source of a highly stable lamp is collected through two lenses and a
toroidal mirror to be focu'sed through a slit. A parabolic mirror deflects the beam through
the slit onto the mirror grating. The desired light wavelength is coordinated, so it is
reflected off its respected grating through an exit slit. This system allows for quick
changes in light wavelengths and a very tight bandwidth ofthose wavelengths.
The captured wavelength is brought to an inverted microscope (Zeiss, Germany) with
a fiber-optic cable to a dicroic mirror. Any light with wavelengths of400 nm or longer is reflected away. The 360 nm or 380 nm light is bent to shine through an objective
optimized for these wavelengths (Neofluar, Zeiss, Germany). This light shines onto a
2 selected cell or region of cells on a coverslip and excites the fura-2 and Ca +-fura-2 within these cells.
The light emission from the fura-2 (~510 nm) shines through the objective to the
dicroic mirror, which reflects wavelengths above 400 nm (such as ~5l 0 and 630 nm)
down a light path, while the wavelengths below 400 nm (such as 360 and 380 nm) are not reflected. The light path is further filtered with a lens to allow only light above 500 nm to
pass. This filtered light of 500 nm or more is converted to voltage either by a
photomultipier or a digital camera.
The photomultipier converts the photons into voltage, which in tum is converted from
analogue to digital by an analogue/digital converter (lTC-16, lnstrutech, USA). The 48 digital information is recorded, manipulated and displayed with computer software (X-
Chart, HEKA, Germany). After correction for the individual background by subtracting the measurements during the dark phase, the ratio ofthe fluorescence intensity at 360 nm to 380 nm is calculated for each light phase.
The digital camera converts the photons to the corresponding pixels on a computer
framegrabber. The pixel information IS recorded, manipulated and displayed with a computer software package (TILLvisION). The software allows for further analysis of multiple cells in the microscope field or ofan individual cell.
In a certain set of experiments, the cells were excited at UV-light wavelengths of 360 nm and 380 nm at a frequency of 20 ms each and 60 ms of dark, while simultaneous the patch-clamp currents were recorded. Osmotic stress changes the intracellular solute concentration and so, changes fura-2 concentration. Consequently, the intensity of the emission at the isosbestic point (360 nm) is changed. Therefore, fura 2 was used to calculate changes in cell volume of intact cells loaded with fura 2 AM in response to an application of extracellular hypertonic or hypotonic solutions. Simultaneously, the cell's
2 intracellular Ca + concentration response to these stressors was measured by examining the ratio offura 2 emission intensity to light of360 nm and 380 nm. 49 3.5. CELL VITALITY STAINING AND MICROSCOPY
In microscopy experiments examining cell morphology, detachment and vitality, cell coated cover slips were placed in individual wells and incubated with 500 ~l media ofthe specific condition. Photos were taken at 40x of the cells within the wells. Morbidity was measured with 30-45 min incubation of cells with the endo-plasma membrane stain,
0.04% trypan-blue dissolved in 0.85% saline. The total number of stained cells was examined by suspending cells with several vigorous pipettings and placing 10 ~l ofthese suspended cells on a hemocytometer at microscopic power of lOx. 50 CHAPTER 4. RESULTS
4.1. TRPM6 AND TRPM7 OSMOSENSITIVITY
4.1.1. Hypertonic-induced Inhibition ofTRPM7 and MagNuM
TRPM7 osmosesnsitivity was explored, because other TRP channels are osmosensitive and, osmotic stress signaling may involve plasma membrane kinases and channels (Strotmann et aI., 2000; Liedtke et aI., 2000; Okada et aI., 2001; Patel &
Honore, 2001; Grimm et aI., 2003; Sheikh-Hamad & Gustin, 2004; Voets et aI., 2004;
Maroto et aI., 2005). Endogenous TRPM7-like currents (MagNuM) and recombinant mouse TRPM7 currents are rapidly inhibited by application of sucrose or NaCl supplemented hypertonic bath solution in a dose-dependent manner, but are not affected by isotonic application (figures 4.1.1 and 4.1.2). TRPM7 and MagNuM currents are proportionally decreased with respect to the characteristic current-voltage (I-V curve) relationship and reversal potential (0 mV) is maintained. This shows TRPM7 channel is hypertonic sensitivity and this hypertonic sensitivity may occur in physiology. 51 A B 400 ~200 310 ~ ' 100 '- ::J U o -30~~~~~ -60+- o 300 600 -1 time [5] C 200 D 400 310 . « , .e. . C 100 ,. Q) « ' '- 200 Q.. ::J U o ------~-- -30~~~~~ 880 -60...1; o 200 400 -1 time [5] Figure 4.1.1. Hypertonic conditions inhibit the endogenous TRPM7-like currents (MagNuM) in non-induced HEK-293s. Mean inward (-80 mY, lower line) and outward 2 (+80 mY, upper line) MagNuM currents develop with 0 Mg + media perfusion and are inhibited with extracellular applications (solid bold line) of A) 480 (n=5), and C) 880 mOsm (n=4). The 1-Y plots display the current inhibition to voltage with applications of B) 480 and D) 880 mOsm. A ~ 2.0 .s +-' c 1.0 ....(J.) ::J () 0.0 -0.1
0 300 time [s] C 2.0 «.s c1.0 Q) .... :J U 0.0 -0.1~~~~~ o 300 time [s]
E 2.0 «c c 1.0 -Q) .... :J U 0.0 -0.1 -0.2-+-----= ri!~~~~ o 300 time [s] G 1 2.0 +-'c ....(J.) 1.0 ::J () 0.0 o -0.10 r--Ill!ItGlI!.li~lIlIlI!IllIIIl!Ill!!IlIO -0.5 -1.0--..-----__------. o 300 600 0 300 600 time [s] time [s] Figure 4.1.2. Hypertonic conditions inhibit TRPM7. Mean inward currents at -80 mY (lower line) and outward currents at +80 mV (upper line) carried by recombinant mouse 2 TRPM7 develop in 0 ATP and 0.9 mM Mg + internal solution and are not affected by 53 extracellular applications (solid bold line) of G) isotonic bath solution (n=8), but are inhibited by hypertonic bath solution with sucrose (mOsm) A) 380 (n=6), C) 480 (n=4), and E) 880 (n=3), or H) with NaCl (580mOsm) (n=3). The current inhibition is in relationship to voltage, shown in the I-V plots just prior (broken line) and at maximal effect (solid line) ofapplications (mOsm): B) 380, D) 480, and F) 880.
Increasing the osmolarity inhibits TRPM7 currents recorded at -80 mV (inward current) and at +80 mV (outward current) in a dose-dependent manner (KD=~430 mOsm). This is illustrated for the average maximal inhibition of mouse TRPM7 current record (the smallest current) in figure 4.1.3A and the percent inhibition of the average mouse TRPM7 current as a function of the log osmolarity of the application solution
(figure 4.1.3B).
A...... 8 0 ~2 ...... c ...... 0 c :;:::; ~ 1 :.0 ...... c -50 °' ••••••••• 0 ::J c () ° o '::!<0 -0.1 -100 2345678 2 234567 2 1 log tonicity [Osm] log tonicity [dsm]
Figure 4.1.3. TRPM7 is inhibited by high osmotic concentrations in a dose dependent manner. The graphs display A) the mean extreme recombinant mouse TRPM7 inhibited currents at -80 (lower trace) and +80 mV (upper trace) and B) the percent inhibition of the average inward (broken line) and outward (solid line) currents just prior to application by those at the end ofapplications of(log mOsm): 200 (n= 13), 320 (n=8), 380 (n=6), 480 (n=4), 880 (n=3) and 2200 (n=4). The box indicates the isotonic range of295-330 mOsm.
At negative potentials, TRPM7 currents are associated with divalent cation-specific influx and increased TRPM7 currents size correlates to an increase in the levels of
2 intracellular free-Ca + (Nadler et aI., 2001; Monteilh-Zoller et aI., 2003). TRPM7 carried
Ca2l influx can be further increased with hyperpolarization to -80 mV. Hypertonic 54 applications simultaneously inhibit TRPM7 carried currents and associated intracellular free-Ca2+ levels. Figure 4.1.4 illustrates the traces from one experiment of four with
similar results, except for intracellular free-Ca2+ ([Ca2+]i) became very large in two experiments after the application was removed. TRPM7-currents and [Ca2+]i increase with perfusion of patch-pipette solutions containing the photoindicator Fura-2 and lacking ATP with periodic 5-s hyperpolarizations to -80 mY, and both simultaneously decrease with hypertonic application. This indicates that TRPM7 divalent influx is also inhibited by hypertonic stress.
600 4~ « 3E. 400 2~.... ~ .... c 1 B ci: 200 0.0 o -0.1 o -0.2 o 300 600 time [5] Figure 4.1.4. Hypertonic conditions simultaneously inhibit TRPM7 currents and influx pathway of Ca2+. Simultaneous whole-cell patch-clamp recordings of TRPM7 carried currents at -80 mY (lower trace line) and +80 mY (upper trace line) and fura-2 measurements of [Ca2+]i (bushy line) developing in ATP-free and I mM Mg2+ filling pipette solution with -100 to +100 mY ramp protocol and periodic 5-s hyperpolarizations to -80 mY. At 300s after whole-cell configuration, a 720 mOsm (solid bar) extracellular solution was applied.
2 Intracellular Mg + negatively regulates TRPM7, but hypertonic inhibition on TRPM7 was not affected by divalent cation concentrations (Nadler et a!., 2001). It rapidly occurs in nominally divalent cation free conditions in the same manner as standard condition
(Figure 4.1.9C and D). When the patch-clamp intracellular and extracellular solutions are devoid of divalent cations and further chelating any other residual divalent cations by 5 55 mM Na2EDTA, then TRPM7 divalent cation-specific influx is removed and the currents increase and become linear associated with the inward and outward monovalent cationic fluxes (Nadler et aI., 200 1; Figure 4.1.9B). The hypertonic induced inhibition of these monovalent cation currents occurs, but is not as rapid or dramatic as any other conditions tested (Figure 4.1.9 A and B). This indicates that TRPM7 inhibition by hyper-osmolarity is not a result of directly changing intracellular divalent cations, but most likely involves at least nominal divalent cations. DVF+EDTA A 20 B 20 ...... ~ 10 nA . c +-'c 0 ....Q) ::J () -10 -20 o 200 400 -20 time [s] DVF c 15 D « - 20 's10 • +-' 15 • c • ~ • lo- 5 • :::J 10 « .• U o c .' I 5 , -3 .• mV .• , o 300 600 .- time [s] -50 50 Figure 4.1.5. Hypertonic inhibition of TRPM7 occurs in nominal divalent cation conditions, but is blunted in conditions devoid of divalent cations. Recombinant mouse TRPM7 carried currents in filling-pipette media devoid of divalent cations with 5 mM Na2EDTA (DVF+EDTA) or nominally free of divalent cations (DVF) display large and linear currents from corresponding extracellular applications of DVF+EDTA and to a lesser extent DVF. Mean inward currents at -80 mV (lower trace) and outward currents 56 at +80 mV (upper trace) carried by TRPM7 are illustrated with A) DVF+EDTA and an 680 mOsm DVF+EDTA application (solid bar) (n=3) and, C) DVF with an 880 mOsm (closed circles, n=4) or 310 mOsm (open circles, n=3) DVF application (solid bar). The I V plots in B) illustrate the large linear TRPM7 currents (broken line) immediately following extracellular application of 670 mOsm DVF+EDTA followed by inhibition of the current size 100s later (solid line). The I-V plots in D) illustrate TRPM7-carried currents in DVF filling-pipette conditions just prior to application (dash-dot-dash line) and those currents immediately following (broken line) and 60 s after (solid line) extracellular application of880 mOsm DVF.
4.1.2. Hypotonic-inducedfacilitation ofTRPM7
2 TRPM7 is involved in cellular Mg + homeostasis and TRPM6 is linked to systemic
2 2 Mg + homeostasis, and Mg + negatively regulates TRPM7 and TRPM6 (Nadler et aI.,
2001; Schlingmann et aI., 2002; Walder et aI., 2002; Schmitz et aI., 2004; Voets et aI.,
2 2004). Hypotonic solution facilitation of TRPM7 requires high Mg + or high Mg'ATP intracellular solutions. There was no substantial effect by hypotonic solution (200 mOsm) on endogenous MagNuM or over-expressing TRPM7 currents when the filling- pipette
2 media contains physiological or low free Mg + (0.9, 0.4 or 0 mM) and lacks Mg ATP
(figure 4.1 .6A and F). This is in agreement with Jiang et aI., 2003, who found that hypotonic applications do not affect microglial MagNuM currents. However, extracellular hypotonic applications (I80 and 200 mOsm) do induce an increase in
2 TRPM7 current when the filling-pipette internal media contains 3mM Mg + (figure
2 4.1.6B and C) or contains physiological 0.9 mM Mg + with 4mM Mg ATP (figure
4.1 .6D and E). Hypotonic stress does not alter TRPM7 currents completely suppressed by
2 ]0 mM Mg + intracellular solution, and slightly or does not alter currents suppressed by 5
2 mM Mg + (data not shown). Balloon-patch-induced cell swelling (see next section) does
2 not affect TRPM7 currents and most probably, Mg + concentrations, in isotonic 57 conditions (data not shown). These results indicate that hypotonic induced facilitation of
2 TRPM7 requires the channel to be partially inhibited by intracellular Mg + or Mg ATP
2 and that the hypotonic effect may act directly by changing local Mg + concentrations.
The hypotonic facilitated currents are proportionally increased with respect to the characteristic TRPM7 current-voltage (I-V curve) relationship while the reversal potential (0 mV) is maintained (figure 4.1.6C and E). This indicates the hypotonic effects are from the cation flux through TRPM7 channels and not other channels, such as stretch sensitive cr and K+ channels. The possible activity of the later channels was suppressed by internal pipette-filling solution containing Cs+ to block K+ channels and 10 mM
2 BAPTA to buffer Ca + to a minimum. In some experiments 100!-!M DIDS (Cr channel antagonist) was added to bath and application solutions to suppress stretch-sensitive cr channels (Oiki et aI., 1995). 58 A B 200 3 ~ 's2 +-'c ~ 1 :::J <..> o -0.1 -0.2T------r------, 300 600 0 300 600 time [s] time [s] D 1.0 200
"EO.5 Q) ..... :::J <">0.0 310 -0.1 ""T------r------, o 300 600 E time [s] 1.0 ~ 200 ...... c "C0.5 Q) ..... 0.5 6 . ~_:mlII_m__~_rl__(C§ll_IllIli__m__1'l_W _ 0 0 310
-0.1 "T------r------, o 300 600 time [s] Figure 4.1.6. Hypotonic conditions facilitate TRPM7 currents partially inhibited by 2 2 intracellular Mg + or Mg ATP, but not internal solutions with 0 or 0.9 mM Mg + and 0 ATP or endogenous TRPM7 MagNuM currents. Whole-cell patch-clamp recordings of mean inward (~80 mV, lower line) and outward (+80 mV, upper line) currents with 200 mOsm extracellular application (solid bold line) carried by A) endogenous TRPM7 2 (MagNuM) in non-induced cells without ATP or Mg + in the filling-pipette media (n=8) 2 or recombinant mouse TRPM7 with filling-pipette media of: B) 0 ATP and 0.9 mM Mg + 2 2 (n=8); C) 0 ATP and 3 mM Mg + (n=6); or E) 4 mM Mg ATP and 0.9 mM Mg + (n=5). Hypotonic (200 mOsm) effect on current-voltage signature of recombinant TRPM7 just prior to application (broken line) and extreme effect (solid line) with an filling-pipette 2 2 media ofD) 0 ATP and 3 mM Mg +, or F) 4 mM Mg ATP and 0.9 mM Mg +. 59 4.1.3. Balloon Patch Counteracts Hypertonic Inhibition ofTRPM7 Current
The 'balloon patch' method applies a pressure to the internal fluid of the filling pipette, which results in the cell swelling like a balloon. The decrease in volume and coinciding inward membrane pressure from hypertonic extracellular fluid can be countered with an opposite pressure applied by the balloon patch, even though the hypertonic application continues. This balloon patch pressure rapidly reverses hypertonic-induced inhibition of TRPM7 currents to pre-application sizes (Figure
4.1.7A). This indicates the hypertonic inhibition ofTRPM7 is not directly by the osmotic gradient or related water efflux, since both still would occur in the "ballooned" cell, but are a result of changes in cell volume or lipid deformation. The balloon-patch readily reversed the hypertonic inhibition of TRPM7 in solutions devoid of divalent cations with
5mM EDTA (Figure 4.1.7B). TRPM7 currents do not increase beyond pre-application sizes after balloon-patch has been applied and immediately decrease from the hypertonic application, when the balloon-patch pressure was removed, even though, the cell volume was visible much larger, than the initial volume. This indicates that the inhibition and the reversal ofthis inhibition are not due to divalent cations or directly from cell volume. 60 A 4 .s« c 2 :::Q) o:::J o -0.1 -0.2 I,.----~:_---__,----=~~~~ -20 r----~--___, o 200 400 600 0 400 800 time [s] time [s] Figure 4.1.7. Balloon patch rapidly reverses hypertonic inhibition, independent of divalent cations. Extracellular 480 mOsm application with A) or B) without divalent cations inhibits recombinant mouse TRPM7-carried inward (-80mV, lower lines) and 2 outward (+80 mV upper lines) current records with internal media ofA) 0.9 mM Mg + or B) EDTA buffered divalent cation-free. The inhibition during application is reversed with by balloon patch pressure (arrows) to pre-application currents sizes, until the balloon patch pressure is removed, then a blunted hypertonic inhibition returns.
4.1.4. Basic pH, Cytochalasin D Nor cAMP Alter Osmotic Effects on TRPM7
TRPM7 current size can be facilitated with intracellular conditions of elevated cAMP and inhibited with agonist-associated depressed intracellular cAMP (Takezawa et aI.,
2004). However, TRPM7 enhanced activity by filling-pipette media with 100 11M cAMP is inhibited by hypertonic applications in a similar manner as control conditions (figure
4.1.8A). This indicates cAMP and cAMP protein kinase are not mediating TRPM7 osmosensitivity.
Disruption of the cytoskeleton with colchicine and cytochalasin D enhanced the mechanosensitivity of the K+ channel, TRAAK, while lowering pH enhanced the mechanosensitivity of TREK-I and TREK-2 single-channel on-cell recordings (Maingret et aI., 1999a; Maingret et aI., 1999b; Bang et aI., 2000). The possibility of the osmotic effects on TRPM7 being due to strain on the membrane-cortical actin cytoskeleton was examined with the f-actin toxin, cytochalasin-D. This treatment swelled and detached the 61 cells, but did not alter the hypertonic or hypotonic effects on TRPM7 currents (figure
4.1.8C & D). TRPM7 is inhibited by low intracellular pH (Gwanyanya et aI., 2004;
Kozak et aI., 2004). However, basic internal solutions (pH 8) had no effect on hypertonic inhibition of TRPM7 currents (figure 4.1.8B). These results indicate TRPM7 osmosensitivity is independent of the pH and the actin cytoskeleton.
A 1.5 B 6 C 5 . 310 D2 310 . 310 • 200 • • nA • • • 4 • • • • 1.0 • 4 • • nA • • nA • nA •• • 3 • • • I 1 • • • • • 2 • .310 • 2 • • 580 0.5 • • • • 480 • • 480 •
-50 50 Figure 4.1.8. Osmotic effects on TRPM7 currents are not altered by intercellular pH or cAMP or actin filament toxin, cytochalasin D (Cy-D). I-V plots are shown of recombinant mouse TRPM7 cells with filling-pipette media of: A) (in mM) 0.1 cAMP, 3 2 2 2 Mg'ATP, 0.4 Mg \ B) pH 8 and 0.9 mM Mg +, and 5 liM Cy-D with C) 0.9 mM Mg + or 2 D) 3 mM Mg + in cells incubated over-night in Cy-D, currents just prior to (broken line) and at maximum effect (solid line) of applications ofA) 480, B) 480, C) 580 and D) 180 mOsm.
4.1.5. Intracellular Osmotic Stress Effects on TRPM7 Currents
Mammals tightly regulate extracellular osmolarity by the kidney and kidney- regulating hormones, such as vasopressin and the adrenal steroids, cortisol and aldosterone (Hardman et aI., 1996). On the other hand, intracellular osmolarity likely fluctuates from the normal metabolic and functional activities of the cell. Therefore, changing intracellular osmolarity is a more likely indicator ofphysiological conditions.
Cells visually observed by a microscope (40x) shrink from hypertonic application and swell from hypotonic application, but there is no noticeable change in capacitance by 62 either hypotonic or hypertonic stressors (data not shown). In a similar, but less pronounced manner, cells in the whole-cell patch-clamp configuration with internal filling-pipette solution that is hyper-osmotic swell and that which is hypo-osmotic shrink.
Hypo-osmotic (230 mOsm) ATP-free filling-pipette solution suppresses TRPM7 currents from developing to the size of iso-osmotic ATP-free currents. Extracellular 230 mOsm application increases the currents to sizes similar to iso-osmotic ATP-free currents
(figure 4.1.9.A). The currents maintain the I-V curve and reversal potential (0 mV) of
TRPM7 (figure 4.1.9.B). Hyper-osmotic (480 mOsm) ATP-free filling-pipette solution in
TRPM7 over-expressing cells develop currents larger than iso-osmotic conditions. These currents are suppressed to iso-osmotic current size by 480 mOsm extracellular applications (figure 4.1.9.C). In certain cells, a monovalent cation current appears in the first 40 s of whole-cell configuration. This current is buffer-subtracted (removed) from the I-V curve with the analysis software (HEKA Patch-Pulse). The application-induced current decrease maintains the I-V relationship and reversal potential (0 mV) characteristics of TRPM7 (figure 4.1.9.D). Hypotonic extracellular fluid does not alter endogenous TRPM7-like, MagNuM currents, but future research needs to examine intracellular hyper-osmotic solutions effect on MagNuM currents.
A well characterized anion current ( lel- stretch) is sensitive to hypotonic extracellular fluid and intracellular ionic strength (Hoffman et aI., 1979 as reported in Grinstein et aI.,
1982; Nilius et al., 1998; for review see Sardini et aI., 2003). both facilitate TRPM7 currents, and hyper-osmotic extracellular and hypo-osmotic intracellular solutions inhibit
TRPM7 currents. The osmotic differences and effects are similar in hypotonic extracellular/ iso-osmotic intracellular solutions and isotonic extracellular/ hyper-osmotic 63 intracellular solutions, but the intracellular ionic strengths are opposite, and the same is true for hypertonic extracellular/ iso-osmotic intracellular solutions and isotonic extracellular/ hypo-osmotic intracellular solutions. This shows the osmotic effects on
TRPM7 are not due to ionic strength.
200 in- & external A B 1.0 « 1.2 c '::' 0.8 c ....~ 0.4 0.5 « ::J c () O.O~~~~~ 200 in- & 310 external -0.1 ~ I -0.2 ~------,-----"If mV , o ... '
D C« 3 5 .s2 480 in- & 310 external ...... 4 • c • •• ~ 1 3 I ::J • () « : 480 in- & external o 2 c -0.2 1 -0.4 -r------T mV o 300 600 time [s] -50 50 Figure 4.1.9. Hypo-osmotic or hyper-osmotic intracellular conditions effect TRPM7 currents and these effects are removed with extracellular application of an equal osmotic strength. Mean inward (at -80 mY, lower graph) and outward (at +80 mY, upper line) currents carried by recombinant mouse TRPM7 with a filling-pipette media of A) 230 (n=7) or C) 480 mOsm (n=4) and an extracellular application of corresponding osmolarity (solid bold bar). The application effects are in a TRPM7 I-V signature manner, as shown in the I-V plots just prior to application (broken line) with filling pipette media of B) 230 and D) 480 mOsm and at extreme application effect (solid line) ofcorresponding osmolarity. 64 4.1.6. Truncated TRPM7 without the Kinase Domain is Hyper-sensitive to Osmotic
Stressors.
TRPM7 contains a cytosolic kinase domain in addition to the transmembrane channel domain (Runnels et a!., 2001; Ryazanova et a!., 2004). HEK-293s over-expressed a truncated TRPM7 without the kinase and succeeding carboxyl-terminal regions (TRPM7 d-kinase) are display a current-voltage relationship identical to that of TRPM7, except
2 TRPM7-d-kinase currents are more sensitive to Mg + negative regulation (Schmitz, et a!.,
2003). Similarly, TRPM7-d-kinase carried currents are hyper-sensitive to hypertonic
(480 mOsm) application-induced inhibition (figure 4.1.10A). Hypotonic application- induced facilitation of TRPM7-d-kinase does not require suppression of currents with
2 Mg + and or Mg'ATP (figure 4.1.10C). The reversal potential (0 mV) and I-V relationship of TRPM7-d-kinase are similar to TRPM7 and inhibition or facilitation of
TRPM7-d-kinase by corresponding extracellular osmotic stressors maintained the reversal potential (0 mV) and I-V relationship (figure 4.1.10B and D). The results imply
2 the inhibitory mechanism of Mg + on TRPM7 channel is similar or identical to the mechanism of TRPM7 channel osmosensitivity, and the sensitivity of this mechanism is regulated by the kinase or the kinase structural support of the rest ofthe TRPM7 protein.
2 Furthermore, these results strengthen the possibility of a relationship between Mg + and/or Mg'ATP concentrations and channel osmosensitivity. 65 A B 3 3 ~ 310 • ...... c • I +-' 2 c I Q.) 2 • ~ • ~ 1 I ::J « I <.> c I 0 I I 1 • I ~0.1 I .• o 300 600 mV .. 480 time [s] -50 50 C o 1.0 250 ~1.5 c ~.O c ~0.5 ~ 0.5 « ::J c 310 <'>0.0 ~~~__io -0.1 mV o 300 600 time [s] Figure 4.1.10. TRPM7-like currents carried by TRPM7 with the kinase domain deleted are hypersensitive to osmotic stressors. Mean inward (at -80 mV, bottom trace) and outward (at +80 mV, upper trace) currents carried by mutated human TRPM7 by the 2 kinase domain being deleted (TRPM7-~-kinase) with filling-pipette free of Mg + are hyper-sensitivity to applications of A) 480 (n=3) or C) 250 mOsm (n=4) than kinase intact TRPM7. TRPM7-~-kinase carried I-V relationship is illustrated just prior to (broken line) and at maximal affect (solid line) of B) 480 and D) 250 mOsm application 2 with nominally free-Mg + internal media.
4. J. 7. TRPM6/7 Osmotic Sensitivities
TRPM6 and TRPM7 proteins associate closely with each other, possibly forming heteromeric channels in the plasma membrane, while TRPM6 does not reach the membrane (Chubanov et aI., 2004). It has been suggested that it is the TRP617 heteromer, which provides TRPM6's physiological actions on Mg2t (re-) absorption. TRPM6 carried
2 Mg + (re-) absorption likely occurs in regions of the body, where a transcellular osmotic 66 gradients would occur. Similar to TRPM7 over-expressing cells, recombinant human
TRPM6 and TRPM7 co-expressed in HEK-293 cells (TRPM6/7) generate currents that
are similarly facilitated by hypotonic applications (200 mOsm), if the currents are
suppressed with 4 mM Mg·ATP (Figure 4.1.11.A & B). TRPM6/7 carried currents are
inhibited by hypertonic applications (480 mOsm) in a manner similar to TRPM7 (Figure
4.1.11.C & D). Similar to TRPM7-~-kinase, currents carried by TRPM7-~-kinase co-
2 expressed with recombinant human TRPM6 with 0 Mg + internal pipette-filling media
are hyper-sensitive to hypertonic 480 mOsm applications (Figure 4.1.11.E).
A 8 ,--, 4.1.8. TRPM7 Over-Expression Does Not Alter Cell Volume-Responses to Osmolarity TRPM7 over-expressing HEK-293 cells regulate volume similar to non-induced cells in response to osmotic stress. The ~ 510 nm fluorescent emission of Fura 2-AM, a non- invasive fluorescent dye made into a polar and active form (Fura-2) by intracellular esterases, excited with 360 nm light, was compared between non-induced and mouse TRPM7 over-expressing cells (24 hours induction) incubated in 5 !-!M Fura 2-AM for 30 min. Hypertonic-induced cell shrinking increases the concentration of dye and thus the light emission intensity, while hypotonic-induced cell swelling dilutes the intracellular dye and decreases the light emission intensity (figure 4.1. 12). The dependent variable on the graphs in figure 4.1.12 is the fluorescence intensity percent decrease from a baseline point (20 s from recording). Therefore, the lower an intensity value is, then the larger the percent-decrease from baseline and visa versa. The graphs were expressed in this manner to associate the intensity changes directly with volume changes. 68 A 0.4 B 0.3 0.2 (]) ~ 0.3 en (1j (1j (]) (]) (:) (:) (]) (]) 0.1 o 0 ~ 0.2 ~ '(j) '(j) c c 0.0 (]) (]) C c ';!( (ft 0.1 0 -0.1 0.0 -0.2 o 100 200 300 400 500 600 0 100 200 300 400 500 600 time [s] timers] Figure 4.1.12. TRPM7 over-expression dose not disrupt cell volume regulation to osmotic stress. Non-induced (closed circles) and 24-hour tetracycline-induced recombinant mouse TRPM7 (open circles) HEK-293s incubated in 5 flM Fura 2-AM show similar mean emission (~51 0 nm) intensity percent-decrease from the record at 20 s in isotonic bath, A) hypertonic (180 mOsm) or B) hypotonic (480 mOsm) conditions (in all conditions, n=3). Fura-2 was excited with 360 nm light. 69 4.2. THE CHANNEL AND THE KINASE INVOLVEMENT IN TRPM7 OVER- EXPRESSION-INDUCED-CHANGES OF CELL MORPHOLOGY 4.2.1. TRPM7 and TRPM617 Over-Expression Changes Cell Adhesion and Morphology HEK-293 fibroblasts over-expressing recombinant mouse TRPM7 or human TRPM7 round, clump and detach from the substrate in a time-dependent manner (as originally observed by Nadler et aI., 2001 and illustrated in figure 4.2.l.A & B). The loss of adhesion and changes in morphological of TRPM7 over-expressing HEK-293s were not due to, or immediately followed by, cell death according to trypan-blue staining of attached and detached cells (figure 4.2.2.B). In this figure, cells treated in a hypertonic media are shown to illustrate an environment known to induce cell death. TRPM7 over- expression changes are not from cells entering into necrotic cell death and are clearly separate from the hypertonic cells that do undergo cell death. The adhesion can be partially rescued with pre-treating the glass cover-Slip with poly-lysine (figure 4.2.2.A). HEK-293s over-expressing recombinant TRPM6 do not noticeably differ from transfected HEK-293s without tetracycline-induction, unless co-expressed with recombinant human TRPM6 and TRPM7, which induce enhanced morphological and adhesion changes ofHEK-293s (figure 4.2.l.C). These results imply TRPM6 proteins are not being expressed on the membrane of HEK-293s. This evidence disagrees with Votes et a1. (2004) claims and supports the findings of Chubanov et a1. (2004), that TRPM6 requires TRPM7 to be brought to and/or incorporated into the membrane. 70 Non-induced 24 hours 48 hours A B C Ic...",'_"'""'....,...,...... _ ...... -.,.. r"__----'"...... ,.,• ._ Figure 4.2.1. Over-expression of human or mouse TRPM7, or over-expression of both human TRPM6 and TRPM7 induces cells to round, swell, clump and detach in a time dependent manner. The 40x micrographs show HEK-293 cells non-induced and, 24 and 48 hours after tetracycline induction of A) recombinant mouse TRPM7, B) recombinant human TRPM7 or C) co-expressed recombinant human TRPM6 and TRPM7. 71 A B 100 90 :!!lao Ql BO- U 70 '0 ." ~60 g 60 '0 CD - ~ 50 .2 'E ." a iii 40 ~ 40· Ql 0"- ffi30 ~20 20 tlOc. a 0 C piC M7 plM7 C M7 C·HiOsm M7·HiOsm Figure 4.2.2. TRPM7 over-expression does not induce cells to die, but to detach, and the detachment can partly rescued by coating the cover slips with poly-lysine. A) The bars indicate the percentage of recombinant mouse TRPM7 over-expressing HEK-293 cells confluent in randomly chosen lOx microscope fields of views on glass cover-slips untreated (M7, n=12) or treated with poly-lysine (pI M7, n=6) or non-induced HEK-293s untreated (C, n=lO) or treated with poly-lysine (pI C, n=4). B) The bars indicate the averaged total cell-counts of dead HEK-293s indicated by trypan-blue stain. Cells were non-induced and incubated in hypertonic 480 mOsm media (C-HiOsm, n=9) or isotonic media (C, n=6) or recombinant mouse TRPM7 over-expressing cells incubated in hypertonic 480 mOsm media (M7-HiOsm, n=9) or isotonic media (M7, n=9). 4.2.2. Ca2+and or Ml+ Media Levels on TRPM7 Over-Expression Effects The primary divalent cations conducted by TRPM7 channel in physiological 2 2 conditions are Ca + and Mg + (Monteilh-Zoller et aI., 2003). Neither non-induced nor TRPM7 over-expressing cells incubated in either high CaCh (11 mM) media (figure 4.2.3.A) or high MgCh (11 mM) media (figure 4.2.3.C) appear to exhibit any differences from cells incubated in standard media. This implies TRPM7 channel over-expression 2 2 induced effects on cells are not the result ofincreased conductance ofMg + or Ca +. 2 Cells without Ca + become round, detach and remain viable in a manner undifferentiated from cells over-expressing TRPM7 (figure 4.2.3.B). The combination of 2 removing Ca + from the media and over-expression of TRPM7 enhanced the rate of the 2 2 changes in cell morphology and adhesion. Low Ca + and low Mg + media 72 indiscriminately kills TRPM7 over-expressing and non-induced cells by 24 hours (Figure 2 4.2.3.D). These results indicate Ca + and TRPM7 act on the same physiological pathway, 2 but the Ca + is not necessarily conducted by TRPM7. Non-induced--...".. 24 hours 48 hours A B o 2 Figure 4.2.3 TRPM7 over-expression and incubation in Ca +-free media induce similar 2 changes in cell morphology and adhesion, and the two conditions are additive. High Ca + 2 or high Mg + media has little to no noticeable affects on non-induced cells or TRPM7 2 2 over-expression-induced changes. Low Mg + and Ca + media is indiscriminately lethal. The 40x micrographs show HEK-293 cells 0, 24 and 48 hours after induction of recombinant mouse TRPM7 incubated in A) II mM CaCh media, B) 10 mM BAPTA 0- 73 2 Ca + media, C) 11 mM MgCh media, and D) 6.8 mM HEDTA buffered media to ~O.l 2 2 mM ofboth Mg + and Ca +. 4.2.3. Kinase Mutations Effects on Cell Adhesion and Morphology In addition to forming divalent-cation specific channels, TRPM7 and TRPM6 form a- kinases. HEK-293 cells over-expressing truncated human TRPM7 lacking the carboxyl- te~inal region including the a-kinase domain (TRPM7-~-kinase), did not exhibit any noticeable phenotypic differences from non-induced cells (figure 4.2.4.C). Unless TRPM7-~-kinase were co-expressed with recombinant human TRPM6, then over- expressing cells did undergo changes in morphological and adhesion (figure 4.2.4.D). Over-expression of recombinant human TRPM6 alone did not induce any noticeable changes in cells (data not shown), but this is likely due to TRPM6 requiring TRPM7 or TRPM7-~-kinase to reach the membrane (Chubanov et aI., 2004). Cells over-expressing single-point mutations of TRPM7 a-kinase domain did exhibit TRPM7 over-expression induced morphology changes (figure 4.2.4.A and B). These mutations of a glycine (1599) to a glutamate in the glycine-rich loop or a lysine to an arginine (1648) in the ATP grasping site are believed to render the kinase phosphotransfer activity disfunctional, while retaining the kinase structural features, such as dimerization (Runnels et aI., 2001; Yamaguchi et aI., 2001; Schmitz et aI., 2004). These results indicate TRPM7 over- expression effects on cell morphology, adhesion and aggregation are due to the a-kinase or the structural stability of the protein provided by the kinase dimerization, but not the kinase phosphorylation activity. 7 Non-induced 24 hours 48 hours 'i5ViE . Figure 4.2.4. Over-expression ofTRPM7 without a kinase does not induce any noticeable morphological changes unless co-expressed with TRPM6 or the kinase is only disabled, but retains structurally integrity. The 40x light micrographs show HEK-293 cells, 0, 24, and 48 hours after tetracycline-induction of single-point mutated human TRPM7 of A) a glycine in the kinase glycine-rich region, B) a lysine in the kinase ATP cleft region, or mutated TRPM7 with the kinase deleted C) without or D) with TRPM6 co-expressed. 4.2.4. Annexin-J Effects on TRPM7 Over-Expression Phenotype TRPM7-kinase binds and activates annexin I by phosphorylating an N-terminal serine (Dorovkov et a1., 2004). TRPM7 over-expression effects occur A) without or with 75 expression of B) annexin I or mutations of the serine of annexin I to C) an alanine to remove the phosphorylation site, or D) an aspartate to mimic the phosphoserine, so the annexin I is always activated. These results indicate TRPM7 over-expression effects on ceIl morphology, aggregation and adhesion are directly involving annexin I. • c Figure 4.2.5. RPM7 ver- pre ion indu e cell to round ell clump and deta h independent of continuous over-expression of activated or inactivated annexin I. The 40x light micrographs show HEK-293 cells, 0, 20, and 48 hours after tetracycline-induction of A) recombinant human TRPM7 alone or with a continuaIly stably expressed B) 76 recombinant human annexin I or single-point mutated human annexin I of a serine in the N-terminal to, C) an alanine (inactivate mutant), or D) a glutamate (continuously active). 4.2.5. MagNuM, TRPM6, TRPM7, TRPM6 + TRPM7, TRPM7-f1-kinase and TRPM6 + TRPM7-f1-kinase Electrophysiological Currents Immediately following whole-cell patch-clamp configuration, voltage ramps were delivered and the corresponding membrane curr~nts were recorded 16 to 28 hours after tetracycline-induction of HEK-293 cells stably transfected with the following recombinant or mutated genes: human TRPM6; human TRPM7 (hTRPM6); mouse TRPM7 (mTRPM7); co-expressed human TRPM6 and TRPM7 (TRPM6/7); single-point mutated human TRPM7 of an a-kinase region glycine (TRPM7-G1599E) or lysine (TRPM7-K1648R); or human truncated TRPM7 mutated to lack the carboxyl-terminal region including the a-kinase (TRPM7-~-kinase);or co-expression of human TRPM6 with TRPM7-~-kinase (TRPM6+M7-~-kinase) (Nadler et aI., 2001; Schmitz et aI., 2004). In similar conditions currents were recorded for transfected HEK-293s without tetracycline-induction (no-tet-HEK) and transiently transfected human TRPM6 (TRPM6- GFP) alone or co-expressed with stably transfected human TRPM7 (TRPM7+M6-GFP) (Voets et aI., 2004). All these channels carry identical current-voltage (I-V) relationships, which represent a divalent cation-specific inward flux and non-specific cation outward flux with a reversal potential near 0 mV (Nadler et aI., 200 I; Monteilh-Zoller et aI., 2003; Schmitz et aI., 2004). In part A of figure 4.2.6, the identical I-V plots are illustrated between TRPM617 and TRPM7, and in part B TRPM6 and no-tet-HEK-293s identical 1- V plots are shown. These results indicate that the channel ion conductance of these different systems were similar. 77 Immunocytochemical staining of the FLAG motif co-expressed with mouse TRPM7 and with human TRPM6, show large expression near the membrane in HEK-293s over- expressing mTRPM7, TRPM617 and TRPM6+M7-~-kinase (Data not shown). These results coupled to the electrophysiological results indicate TRPM6 requires TRPM7 to form a membrane channel in these cells. A B 2 1.0 M6-GFP M6-stable M6-GFP mV M6-stable Figure 4.2.6. TRPM617, TRPM617-GFP and TRPM7 carry identical current to voltage (I V) relationships, and TRPM6, TRPM6-GFP and no-tet-HEK carry identical I-V relationships and current sizes. A) The superimposed I-V plots are taken at a current size of I nA at +80 mV ofHEK-293 cells stably transfected with recombinant human TRPM7 alone (n=4), stably co-transfected with recombinant human TRPM7 and TRPM6 (n=46) and co-transfected with stable TRPM7 and transient TRPM6 (n=5). B) The superimposed I-V plots are after a stable current size is reached (~300s) carried by HEK-293s stably transfected with recombinant human TRPM6 (M6-stable) (n=2) and transiently transfected with human TRPM6 (M6-GFP) (n=3) and a non-induced MagNuM current (broken line). The currents carried by these channels develop for up to 250s before a stable size is reached, depending on the rate of diffusion of cytosolic materials out of the cell, rate of 2 filling-pipette media perfusion into the cell, free-Mg +, Mg·ATP, pH and or osmolarity of the solution (Nadler et aI., 200 I; Hermosa et aI., 2002; Schmitz et aI., 2004; Voets et aI., 78 2004; Gwanyanya et aI., 2004). For each ramp pulse the current sizes at -80 and +80 mV are recorded to correspond with the inward divalent and outward non-specific cation fluxes, respectively, in relationship to time from the onset ofwhole-cell configuration. MagNuM (Magnesium Nucleotide Metal), the endogenous TRPM7-associated current in wt-HEK and no-tet-HEK, is relatively the same size as the currents of TRPM6 and TRPM6-GFP cells (Figure 4.2.6.B). TRPM7 maximum current size corresponding to +80 mV is considerably larger than MagNuM or TRPM6, similar to G1599E or TK1648R currents published by Schmitz, et aI., (2004), and smaller than TRPM7/6. It should be noted that the over-expressed current sizes could be due to clonal differences (certain clones may express more proteins) and induction time. TRPM7-i1-kinase and TRPM6+M7-i1-kinase current sizes are smaller and more sensitive to intracellular free- 2 Mg + negative regulation than TRPM7 or Schmitz, et aI., (2004), published current records ofTRPM7-G1599E or K1648R (Figure 4.2.7). These results indicate TRPM7-i1- 2 kinase and TRPM6+M7-i1-kinase are similarly hyper-sensitive to intracellular Mg +. 79 A B c K1648R G1799D 5.0 5.0 5.0 qlllW:oMg2+ ~2.5 qllTit_b3Mg2+ ~2.5 3 Mg2+ 3Mg2+ =;;~;5Mg2+ ~=;:::::;=~5Mg2+2 1~~~~~~5Mg2" 0.0 ::: 8Mg2+ 0.0 1.,. 8 Mg + 0.0 1.; aMg2+ o 100 200 300 o 100 200 300 o 100 200 300 Time (sec) Time (sec) Time (sec) D E F 1.0 TRPM7-A-Kinase TRPM6n TRPM6n-A-Kinase 2.5 « 2.0 1.5 .s 1.5 c « 1.0 ~ 1.0 .s 1 Mg2,+ ~ ~:~ ------0.0 2 '-t---r----r---. 3 Mg + -0.1 -e~~~.-mi' o 100 200 300 0 100 200 300 0 100 200 300 Time (sec) time [s] time [s] Figure 4.2.7 Currents carried by a kinase-deleted mutant ofTRPM7 are hyper-sensitive to 2 Mg + with or without co-expression of TRPM6. Mean outward currents at +80 mV 2 develop in 0 ATP and 0, 3, 5 and 8 mM Mg + internal solution carried by A) recombinant human TRPM7, single-point mutations in the TRPM7-kinase domain of B) a lysine or C) a glycine, D) mutated human TRPM7 not expressing the kinase domain as published in Schmitz et aI., 2004. Mean inward currents (at -80 mV, lower trace) and outward currents (at +80 mV, upper trace) carried by co-expressed E) recombinant human 2 TRPM6 with TRPM7 and 1 mM Mg + filling-pipette solution (n=6), and F) mutated human TRPM7 not expressing the kinase domain with recombinant human TRPM6 in 2 2 nominally free Mg + (open circles, n=4) and 1 mM Mg + (filled circles, n=3) solutions. 4.3. GLUTAMATE FACILITATES OUTWARD, BUT NOT INWARD TRPM7 CURRENTS The aqueous glutamate zwitterion contains negatively charged carboxyl moieties at the a and y positions and a positively charged amino moiety at the a position. Water and aqueous ions interact with all three charged moieties, but the net charge is 1+. Application of extracellular Na-glutamate media (NaGlu) replacing NaCl media increases TRPM7 or endogenous TRPM7-like MagNuM currents at positive potentials, but not at 80 negative potentials (figure 4.3.1). At physiologically relevant levels of less than 1 mM NaGlu, there are no effects on TRPM7 currents (data not shown). A 8 c .-.3 6 -()8'~ 2 - .2 - .3 o 300 600 -1 time [s] -50 50 D E ~3 3 's2...... c 2 nA ....~ 1 ():::J 0 1 -0.1 o 300 600 time [s] -50 50 Figure 4.3.1. Extracellular sodium glutamate (NaGlu) facilitates TRPM7 outward currents. Mean outward (+80 mV, upper line), but not inward (at -80 mV, lower line) 2 currents carried by recombinant mouse TRPM7 with 0.9 mM Mg + filling-pipette media are facilitated by extracellular application (solid bold line) of A) 140 mM NaGlu and NaCl-free or D) 70 mM NaGlu and 70 mM NaCl. The I-V curves illustrate NaGlu applications of B & C) 140 mM NaGlu, NaCl-free or D) 70 mM NaGlu and NaCl facilitate the currents associated with positive voltages, but not negative potentials carried by B & D) recombinant mouse TRPM7 or C) endogenous TRPM7-like MagNuM in 0.9 2 mM Mg -+ filling-pipette media, just prior to (broken line) and at maximum effect (solid line) of application and the difference between the two (dashed line). The extracellular NaGlu facilitation of TRPM7 outward currents occurs in a similar 2 2 manner with either Ca -+ or Mg + absent from the application and bath media (figure 2 4.3.2A, B, C & D), but NaGlu effects on MagNuM currents are abolished when both Ca + 2 and Mg + are absent from the application and bath solutions (figure 4.3.2.E & F). 81 A 8 .-20 ~.15 1:10 The inhibition of TRPM7 currents by hypertonic solutions and NaGlu facilitation of TRPM7 currents occur simultaneously and independent from each other (figure 4.3.3). It should be noted that the increased average inward currents exhibited after application in figure 4.3.3A do not appear to be associated with TRPM7. .-.8 .-.10 « ~ 8 AE..6 c4 B :;:' 6 Q) a5 4 t::2 .... :::J :5 2 ()O () 0 ------.... ------0.4 -0.2 -0.4 -0.8 """'I,------f o 300 600 0 300 600 time [s] time [s] Figure 4.3.3. Extracellular hypertonic inhibition and sodium glutamate (NaGlu) facilitation ofTRPM7 carried currents occur simultaneous and independent ofeach other. The mean outward (+80 mY, upper line), but not inward (at -80 mY, lower line) currents carried by recombinant mouse TRPM7 in 0.9 mM filling-pipette media and extracellular 480 mOsm hypertonic application of A) 140 mM NaGlu and 140 mM NaCl or B) 280 mM NaGlu and less than 5 mM NaCl. 83 CHAPTER 5. DISCUSSION TRPM7 and TRPM6 are unique proteins that form both transmembrane channels and intrinsic cytosolic a-kinases, as homomers or as heteromers with each other (Nadler et aI., 2001; Runnels et aI., 2001; Yamaguchi et aI., 2001; Chubanov et aI., 2004). At 2 2 negative"potentials, the channels specifically conduct Mg +, Ca + and trace divalent cations (Nadler et aI., 2001; Monteilh-Zoller et aI., 2003; Voets et aI., 2004). This specificity is theorized to be due to a divalent cation permeation block (Kerschbaum et aI., 2002). At positive potentials, the channels predominantly carry a monovalent cation efflux (Nadler et aI., 2001). However, at low positive potentials, some of the channels appear to be carrying a slight divalent cation-specific influx. Replacing the chloride ofthe extracellular solution with glutamate disruptes this divalent cation-specific influx. 21 Mg , Mg·ATP, PIP2, pH and Gi-coupled transmembrane receptor agonists negatively regulate TRPM7 and/or TRPM6 channel activity, while cAMP and Gs-coupled transmembrane receptor agonists facilitate TRPM7 channel activity (Nadler et aI., 2001; Runnels et aI., 2002; Gwanyanya et aI., 2004; Kozak et aI., 2004; Takezawa et aI., 2004). 2 TRPM7 and TRPM617 channel activity and corresponding intracellular Ca + levels are inhibited by hyper-osmotic (hypertonic) extracellular fluid in a dose dependent manner, while hypo-osmotic (hypotonic) extracellular solutions increase TRPM7 and TRPM617 channel activity. Osmotic differences result in changes in cell volume, membrane stretch, membrane strain and membrane deformation, osmotic pressure upon the membrane, changes in intracellular ionic strength, molecule concentration and molecular crowding. The stimulus responsible for TRPM7 osmosesnsitivty was explored. 84 Osmolarity stress alters cell volume and subsequent membrane strain, as well as providing a direct pressure onto the membrane. Therefore, cells need to maintain equal intracellular and extracellular osmolarity. TRPM7 channel and/or kinase osmosensitivity may provide an intracellular signaling mechanism ofosmotic stress. TRPM7 a-kinase phosphorylates serines and threonines within a-helical secondary structures of TRPM7, annexin 1 and possibly other proteins (Runnels et aI, 2001; Yamaguchi et aI., 2001; Dorovkov et aI., 2004; Langes1ang et aI., 2004; Ryazanova et aI, 2004; Schmitz et aI., 2004). Phospholipase Cy is found to be associating with TRPM7, but the exact interaction is not known (Runnels et aI., 2001). Cells over-expressing TRPM7 become round, swell, clump and detach. The over-expression effects can be prevented by removing the kinase, as was seen in cells over-expressing truncated TRPM7 with the kinase region not expressed. Neither, the channel activity, the kinase activity or annexin 1 appeared to influence these effects. This suggests the kinases structure, but not necessarily phosphorylation activity binds and thereby inactivates a protein involved in 2 cell morphology, adhesion and aggregation. Cells incubated in media devoid of Ca + 2 mimicked TRPM7 over-expression effects, this suggests Ca + could be involved in this proteins actions. Models and explanations are discussed of possible novel TRPM7 characteristics and physiological functions based on literature review and complemented with the experimental results of the etiology of TRPM7 and TRPM617 over-expression effects, TRPM7 regulation of cell volume, TRPM7 and TRPM617 osmosensitivity and extracellular glutamate effect on TRPM7 divalent cation permeation block. 85 5.1. TRPM7 CHANNEL IS OSMOSENSITIVITY 2 5.1.1. Hypotonic Facilitation ofTRPM7 as an Intracellular Ca + Signal The ability of many mammalian cells to adjust to hypotonic induced swelling and associated membrane stretching is largely dependent upon an initial influx of 2 2 extracellular Ca + into the cytosol. The actual Ca + pathways responding to hypotonic stress or associated cellular changes has'yet to be proven and clarified (for review see 2 Okada et aI., 2001). It should be noted, this study did not observe intracellular Ca + changes in re~ponse to hypotonic stress. 2 TRPM7 channel facilitation by hypotonic stress likely increases intracellular Ca +. 2 However, TRPM7 over-expressing cells do not show any difference in intracellular Ca + levels in response to hypotonic stress compared to non-induced cells, and endogenous TRPM7-like MagNuM currents are not altered by hypotonic stress. These results suggest 2 TRPM7 is not the Ca + pathway ofthe hypotonic stress signal. In addition, TRPM7 over expression does not effect the volume readjustment to osmotic stressors. Cells over expressing TRPM7 have identical changes in cellular volume to non-induced cells in response to either hypertonic or hypotonic stress. TRPM7 over-expressing cells undergo morphological changes similar to hypotonic stressed cells and cells treated with cytochalasin D. However, incubating TRPM7 over expressing cells in hypertonic media did not noticeably delay these changes and vice versa, TRPM7 over-expression does not rescue hypertonic media-induced cell death. 86 5.1.2. Intracellular Signaling ofHypertonic Stress Hypertonic solutions could be inhibiting TRPM7-kinase activity, in addition or because of hypertonic inhibition of TRPM7-channel. In yeast, extracellular hypertonic stress inhibits membrane-bound kinases, such as SIn I, in a dose-dependent manner. Thereby, removing these membrane-bound kinases inhibition of the high osmotic glycerol (HOG) MAP kinase signal transduction c'ascade, which results m gene expression (Brewster & Gustin, 1994; Maeda et aI., 1994; Gustin et aI., 1998; Hohmann, 2002; Kliltz & Burg, 1998). In C. elegans a MAP kinase pathway is used to signal hypertonic extracellular stress and initiates behavioral and physiological responses (Solomon et aI., 2004). Perhaps, TRPM7-kinase activity represses the putative mammalian analogue to the yeast signal transduction cascade of hypertonic stress activated MAP kinases (for review see Sheikh-Hamad, et aI., 2005). Hypertonic stress leads to the nuclear translocation of a transcription factor NFAT5, also known as TonEBP (for review see Iqbal & Zaidi, 2005). NFAT5 expression or nuclear translocation could be regulated directly or indirectly by TRPM7 and vice versa NFAT5 could regulate TRPM7 expression. 5.1.3. Gating-Mechanism There are many possible stimuli, which could be responsible for TRPM7 and TRPM617 osmosensitivity. Osmotic differences result in changes in cell volume, membrane stretch, membrane strain and membrane deformation, osmotic pressure upon the membrane, changes in intracellular ionic strength, molecule concentration and molecular crowding. Many mammalian channels sense osmotic stressors or other factors 87 inflicting pressure to the cell membrane and or changing cell volume, membrane strain and ionic strength (for reviews see Sardini et aI., 2003; Patel et aI., 2001; van der Wijk et aI., 2000). Many K+ channels are directly or indirectly osmosensitive, including TRAAK, TREK- 1 and TREK-2 (Maingret et aI., 1999a; Maingret et aI., 1999b; Bang et aI., 2000). Disruption of the cytoskeleton with colchicine and cytochalasin b enhanced the mechanosensitivity of TRAAK, while lowering pH enhanced the mechanosensitivity of TREK-l and TREK-2 (Maingret et aI., 1999a; Maingret et aI., 1999b; Bang et aI., 2000). TRPM7 is also pH sensitive (Gwanyanya et aI., 2004; Kozak et aI., 2004). TRPM7 current size can be facilitated with intracellular conditions of elevated cAMP and inhibited with agonist-associated depressed intracellular cAMP (Takezawa et aI., 2004). However, TRPM7 osmosensitivity is independent ofpH, cytochalasin D and cAMP. The stretch-activated chloride currents (Ic]- stretch) are sensitive to hypotonic extracellular fluid and intracellular ionic strength (Hoffman et aI., 1979 as reported in Grinstein et aI., 1982; Nilius et aI., 1998; for review see Sardini et aI., 2003). Hypo- osmotic extracellular and hyper-osmotic intracellular solutions both facilitate TRPM7 currents, and hyper-osmotic extracellular and hypo-osmotic intracellular solutions inhibit TRPM7 currents. The osmotic differences are similar in the corresponding inhibiting or facilitating stimuli, but the intracellular ionic strengths are opposite. This shows that the osmotic effects on TRPM7 are not due to ionic strength. Some ofthe TRP channel relatives of TRPM6 and TRPM7 are also osmosensitve and or mechanosensitive. NOMPC (or TRPN) and human TRPP2 and TRPP3 are a putative mechanosensitive channels, although there is no electrophysiological evidence and 88 NOMPC is not found in humans (from a review by Huang, 2004). C. elegans OSM-9 is a TRP channel that senses hypotonic stress (Colbert et aI., 1997). TRPV4, a hypotonic sensitive mammalian channel, is similar enough to OSM-9, it can substitute for OSM-9 in C. elgans and rescue OSM-9-knock-out C. elgans sensitivity to hypotonic stressful environment (Strotmann et aI., 2000; Liedtke et aI., 2000; Liedtke et aI., 2003). In 2 2 humans, TRPV4 mechanosensitivity and insuing Ca +, Mg + and monovalent cation influx has been implicated in the circumventricular organs negative regulation of the hypothalamic-posterior pituitary-renal axis regulating systemic tonicity, inner ear hair cell defection and sensory nerve pressure-touch sensation (Liedtke et aI., 2000; Mizuno et aI., 2003; Suzuki et aI., 2003). In the kidney, TRPV4 is distributed in regions impermeable to water and most probably experiencing transcellular osmotic differences, such as the distal convoluted tubule (DCT) (Tian et aI., 2004). Interestingly, TRPM6 expression is limited to the DCT (Voets et aI., 2004). Defective TRPV4 has been implicated in cystic fibrosis lung cell swelling and subsequent airway restriction (Amiges et aI., 2004). Hypotonic activation of TRPV4 has been shown to be VIa a tyrosin kinase phosphorylation or phospholipase Ardependent, 5',6'-epoxyeicosatrienoic acid (Xu et aI., 200; Vriens et aI., 2004). TRPM7 has three tryosin kinase phosphorylation sites, while TRPM6 does not have any (appendix 11). A channel with high homology to TRPM7 and TRPM6, TRPM3, is also sensitive to hypotonic stress (figure 2.3) (Grimm et aI., 2003; 2 Lee et aI., 2003). TRPM3 associated intracellular Ca + was correlated with increases in volume (Grimm, 2003). Similar to TRPV4, TRPM3 is also are gated by a lipid derivative, D-erythro-sphingosine and has been suggested to be involved in systemic tonicity 89 regulation (Lee et aI., 2003; Grimm et aI., 2004). TRPM7 could be regulated by changes in cell volume and or associated changes in intracellular concentrations. Osmotic stress- induced changes in cell volume occur in a parallel manner to the changes of TRPM7 currents. Future research will have to examine if tyrosine kinases, phospholipase A2, other lipases, or lipid derivatives, such as 5',6'-epoxyeicosatrienoic acid or D-erythro- sphingosine, have any influence on TRPM7 and TRPM617 osmosensitivity, as well as, 2 the possibility of these proteins and Mg + being involved in systemic tonicity or vestibulocochlear sensations. 2 TRPM7 is involved in cellular Mg + homeostasis and TRPM6 is linked to systemic 2 2 Mg + homeostasis, and Mg + negatively regulates TRPM7 and TRPM6 (Nadler et aI., 2001; Schlingmann et aI., 2002; Walder et aI., 2002; Schmitz et aI., 2004; Voets et aI., 2004). It is conceivable that changes in cell volume by osmotic stress could change local 2 concentrations of Mg + and Mg·ATP. Hypertonic-induced cell shrinking would raise 2 intracellular Mg + concentration and thereby inhibit TRPM7, while hypotonic-induced 2 cell swelling would lower intracellular Mg + concentration and facilitate TRPM7. This could explain why extracellular hypotonic solution facilitation ofTRPM7 occurs at 3 mM 2 2 Mg + or 4 mM Mg·ATP intracellular solutions, but is absent in very high Mg + (IO mM) 2 2 or low Mg + or ATP. Furthermore, a hypotonic dilution of Mg + concentration explains why swelling cells by applying pressure through the whole-cell recording-pipette, does not change TRPM7 currents. However, the dilution hypothesis does not address, that hypotonic extracellular 2 solution facilitation of TRPM7 will occur without Mg + and ATP, if the currents are suppressed by hypo-osmotic intracellular solution, or when TRPM7 is truncated by 90 removing the kinase domain. Furthermore, hypertonic inhibition of TRPM7 is not affected by divalent cation concentrations. Hypertonic inhibition occurred with the same 2 2 kinetics in conditions with internal and external solutions containing Mg + and/or Ca + or nominally free of divalent cations. Although it should be noted, when the intracellular and extracellular solutions are absolutely devoid of divalent cations by EDTA, the hypertonic-induced inhibition ofthe monovalent cation current is not as dramatic as with nominally divalent cation free or standard conditions. Mg·ATP-inhibition ofTRPM7 also is blunted or removed when EDTA is added to the internal solution (Kozak & Cahalan, 2 2 2003). There are several motifs on TRPM7 suggesting the interaction of Zn +, Ca + and 2 Mg + with the protein, including E-F hand motifs and a Zn finger (appendix II and III). EDTA chelation activity could be interfering with the cations binding at these sites. The hypertonic inhibition of TRPM7 in standard conditions or in conditions devoid of divalent cations with EDTA are both immediately reversed by countering the osmotic- pressure with a pressure applied through the recording-pipette and thereby "ballooning" the cell. This indicates the hypertonic inhibition ofTRPM7 is not directly by the osmotic gradient or related water efflux, since both still would occur in the "ballooned" cell, but are a result of changes in cell volume or lipid deformation. TRPM7 currents do not increase beyond pre-application sizes after balloon-patch has been applied and immediately decrease from the hypertonic application, when the balloon-patch pressure was removed, even though, the cell volume was visibly much larger, than the initial volume. This indicates that the inhibition and the reversal of this inhibition are not due to divalent cations or directly from cell volume. 91 It could be possible, that proteins, phospholipids, intracellular stores and or other 2 2 sources of Mg + or Mg·ATP, release Mg + or Mg·ATP in response to hypertonic stress 2 and remove Mg + or Mg·ATP from the cytosol in response to hypotonic stress. This 2 osmotic stress-induced intracellular Mg + would regulate TRPM7. Mg·ATP levels are believed to change as part of the cellular osmotic stress response (from by Okada et aI., 2 2001). Mg + has yet to be not shown to flux with osmotic stress, but this could a reflection of available technology (for review see Wolf et aI., 2003). Increasing 2 2 intracellular Mg + in response to hypertonic stress and decreasing intracellular Mg + in response hypotonic stress could help in the osmolyte balances across the membrane. However, this scenario cannot explain the rapid reversal ofTRPM7 hypertonic inhibition by balloon patch or why hypotonic solutions do not facilitate endogenous TRPM7-like 2 MagNuM currents in low or nominally free Mg + and Mg- ATP. A possible theory of the cell volume changing an unknown inhibitory factor needs to 2 answer those questions with the Mg + dilution hypothesis. Additionally, it cannot explain 2 the lack of a TRPM7 current response to hypotonic solutions with 0 or 0.9 mM Mg +. 2 Even if Mg + is not directly responsible for the actions of osmotic stress on TRPM7, the two cooperate and may facilitate the actions of the other. Hypo-osmotic stress appears to 2 shift Mg + dose-response curve on TRPM7 current size. The protein mechanism of 2 inhibition could be the same for hyper-osmotic stress, Mg + and/or Mg·ATP. TRPM7 a- kinase appears to similarly regulate the mechanism involved in TRPM7-channel 2 hypertonic inhibition, hypotonic facilitation and intracellular Mg + negative regulation and is involved in the cAMP effects (Schmitz et aI., 2004; Takezawa et aI., 2004). Currents carried by TRPM7 channel without the kinase, are hypersensitive to hypertonic 92 2 inhibition and do not require Mg + suppression for hypotonic facilitation. This implies that the kinase has a regulatory role on TRPM7 osmosensitivity, possibly by setting the 2 2 sensitivity of the channel to Mg + and/or Mg·ATP. Low or lack of intracellular Mg + or ATP activates TRPM7 channel, possibly this activation of the channel activates the kinase regulation of the osmotic sensor of TRPM7. Future research is needed to further 2 understand the relationship between osmolarity, Mg + and Mg·ATP on TRPM7 and in general cellular physiology. TRPM7 osmosensitivity could be via direct mechanical pressure on the channel protein or surrounding lipid membrane. The results of on-cell and inside-out recordings with direct pressure application suggest another TRP channel, TRPC1, is gated by plasma membrane strain. TRPC1 is a mechanosensitive cation channel similar to the endogenous mechanosensitive cation channel of the frog oocyte (Maroto et aI., 2005). As of yet, TRPM7 mechanosensitivity in the single-channel configuration has yet to be recorded. This method would allow for further clarity if TRPM7 osmosensitivity is the result of direct regulation by lipid deformations like TRPCI and the osmosensitive K+ channels TRAAK, TREK-I and TREK-2. However, a mechanosesnitive channel should be sensitive membrane stretch by balloon patch and not require Mg2+ or Mg· ATP suppression ofthe channel. This is not the case. Perhaps, the hypertonic inhibition of the constituatively active TRPM7 is the more physiological relevant effect. Hopefully, future research will uncover the physiological role of TRPM7 osmosenstivity and if the gating mechanism is directly by mechanical stimuli or indirectly by an agonist, such as lipid derivatives like TRPM3 and TRPV4, or phosphorylations like TRPV4, or by an intracellular inhibitory factor, such as Mg2+or Mg·ATP. 93 5.2. TRPM7 a-KINASE IS INVOLVED IN CELL MORPHOLOGY AND ADHESION TRPM7 over-expression with or without co-expression of TRPM6 induces the cells to increasingly become round, swell, clump and detach (Nadler et aI., 200 I). Cells entering into necrosis undergo cell swelling and loss adhesion, but TRPM7 over-expression- induced changes are neither the result of nor immediately followed by cell death. Pre- treating the glass cover-slips with poly-L-Iysine partially rescues cell adhesion and there is actually a lower percentage of dead cells on the cover-slips with this treatment than untreated cover slips (figure 4.2.2). The results showed that the changes in cell adhesion, 2 aggregation and morphology were very similar to those of cells incubated in Ca + free media and were from over-expression of the a-kinase and not increased divalent cation conductance from channel over-expression. TRPM7 over-expression induced changes III cell morphology, adhesion and aggregation were absent in cells over-expressing the truncated TRPM7 with the kinase domain removed (TRPM7-~-kinase) or in cells over-expressing TRPM6, unless the two proteins were co-expressed. According to Voets et a1. (2004) over-expression of TRPM6 reveals a similar current-voltage relationship and currents sizes to and over-expression of TRPM7. However, these findings could not be confirmed in this study. The currents of cells over-expressing TRPM6 were not larger than the endogenous MagNuM TRPM7- like currents in non-induced cells (figure 4.2.6.B). Only cells over-expressing both TRPM6 and TRPM7 (TRPM6!7) exhibited current sizes beyond MagNuM (figure 4.2.2.C). These results support the immunocytochemical and fluroscent resonance emittance topographic (FRET) findings of Chubanov et a1. (2004); that TRPM6 does not 94 incorporate in or near the plasma membrane of cultured cells, unless TRPM6 is co- expressed with TRPM7 (or TRPM7-~-kinase). TRPM7-~-kinase currents are 2 hypersensitive to intracellular Mg + (Schmitz et aI., 2004) and so are currents carried by TRPM7-~-kinase co-expressed with TRPM6 (figure 4.2.7). This implies that the a- 2 kinase and not the Mg + sensitivity of the channel is involved with cell morphology and adhesion. 2 2 Furthermore, increasing Mg + or Ca + in the cell incubation media did not hasten the onset or alter TRPM7 over-expression effects on cells (figure 4.2.3.A & C). It should be 2 noted that increased extracellular Mg + does not necessarily correlate with increased 2 intracellular free-Mg + (Wolf, 2003). However, if the TRPM7 over-expression induced 2 2 phenotype is due to high Mg + or high Ca + conductance, then, conceivably, it would be 2 enhanced by an increase of extracellular Mg +, and this is not the case. The media did 2 2 contain albumin, which may have buffered the additional extracellular Mg + and Ca + 2 (Benet et aI., 1996). High intracellular Ca + has many morphological effects on cells, but these are not the same as those observed in TRPM7 over-expressing cells. Apparently, TRPM7 over-expression effects are fram the TRPM7-kinase binding and thereby inactivating proteins involved in cell morphology and adhesion. The phosphorylation activity is not necessary to bind and inactivate these pratins, because cells over-expressing TRPM7 with single-point mutants of the a-kinase domain induce the over-expression changes of cells (figure 4.2.4.A and B). These mutants disrupt TRPM7-kinase activity, but retain kinase structure (Runnels et aI., 200]; Yamaguchi et aI., 2001; Schmitz et aI., 2004). The mechanism by which over-expression of TRPM7 95 kinase-induces the changes, could be from the kinase directly binding the proteins involved in morphology and adhesion. Alternately, the mechanism could be from the kinase domain dimer formation providing structural stability to the binding site of the proteins involved in morphology and adhesion. There are few TRPM7 proteins expressed III the plasma membrane under physiological conditions (Hermosura et al., 2002). Thus the over-expression induced changes in cell adhesion, aggregation and morphology are likely non-physiological. But, the over-expression induced changes could relate to physiological functions of TRPM7. Maybe, the a-kinase removes proteins from being involved in cell morphology and adhesion. The changes in cell morphology and adhesion are similar to the changes observed with disruption ofthe actin cytoskeleton by the f-actin toxin, cytochalasin-D. Annexin 1 is the only protein known to be phosphorylated by TRPM7 a-kinase, other than itself (Dorovkov & Ryazanov, 2004). Annexin I interacts with the plasma membrane and is implicated in cell adhesion, aggregation and morphology (Roviezzo et al., 2002). Nevertheless, annexin lover-expression shows no effects on TRPM7 over-expression induced changes in cell adhesion, aggregation and morphology (figure 4.2.5). Dictyostelium a-kinases phosphoregulate the a-helical tails of myosin proteins (for review see De la Roche & Cote, 2001). Dictyostelium myosin-Ia motility is regulated by 2 the phosphorylation of the tail regions and in vitro, changes in the free Mg + (Fujita- Becker et al., 2005). TRPM7 a-kinase has been suggested to associate with myosins (Langeslang et al., 2004). Myosin II regulates the tension between the cortical actin cytoskeleton and the membrane (Lodish et al., 2000). This role ofmyosin II appears to be 96 regulated by the phosphorylation state of the heavy chain tails and the calmodulin light chain (Laevsky & Knecht, 2003). TRPM7 may bring myosin II to the plasma membrane and adjust membrane-TRPM7-myosin-actin tension by TRPM7 a-kinase 2 phosphorylating myosin 11 heavy chain tail regions, by TRPM7 conducted-Ca + 2 regulation of the calmodulin and by TRPM7 conducted-Mg + regulating the myosin ATPase activity. One of the ways TRPM7 was originally discovered was by its association with Phospholipase Cy (PLCy) (Runnels et aI., 2001). Perhaps, over-expression of TRPM7 binds-up and thereby inactivates the cell's PLCy. PLCy hydrolyzes PIP2 glycerol- phosphoester bond with inositol 1,4,5-triphosphate (lP3). Both the freed IP3 and the remaining sn-l ,2,-diacylglycerol have actions as second messengers. IP3 stimulates the 2 release of Ca + from intracellular stores, such as the endoplasmic reticulum. This could 2 explain TRPM7 over-expression and incubation media devoid of Ca + similar effects on cell morphology and adhesion. Over-expression ofTRPM7 would bind and inactivate the 2 cell's PLCy, thereby decreasing the IP3-induced release of Ca + from the intracellular 2 2 stores into the cytosol. Removing extracellular Ca + would deplete Ca + from the 2 intracellular stores, thus there would be no Ca + to be released by IP3. TRPM7 and TRPM617 channels are sensitive to changes in osmolarity and the a- kinases appears to be involved with the interaction between the plasma membrane and the cortical cytoskeleton, such as cell morphology, adhesion, and aggregation. These phenomena could be interconnected, such as a cellular way of sensing and adjusting membrane tension. TRPM7 and TRPM617 may have a physiological role of sensing and 97 signaling membrane strain and initiating the rearrangement of the cytoskeleton and other 2 adaptations associated with membrane strain (Di Ciano et aI., 2002). Mg + facilitates TRPM7 a-kinase activity (Ryazanova et aI., 2004). A physiological function of TRPM7 2 is in cellular Mg + homeostasis and the kinase adjusts TRPM7-channel sensitivity to 2 2 intracellular Mg + (Schmitz et aI., 2004). It is possible, that Mg + conducted by TRPM7 channel regulates TRPM7-kinase activity. Based on the results a hypothesis ofan additional physiological function of TRPM7 is that: 1) membrane strain or associated stimuli regulate the opening and closing of 2 TRPM7-channel and its divalent cation conductance; 2) the Mg + influx regulates TRPM7-kinase; 3) TRPM7-kinase phosphoregulates proteins involved in adapting and signaling membrane tension; 4) and the kinase adjusts the channel sensitivity to turgor, as 2 it does to Mg +. Most probably the osmolarity directly or indirectly cooperates with 2 intracellular Mg + and Mg·ATP regulation of TRPM7 and the osmotic regulation of 2 2 TRPM7 is involved with other physiological roles of TRPM7 channel in Mg +, Ca + and trace divalent cations conduction, and TRPM7 a-kinase phosphoregulation of TRPM7 and other proteins. The suggested mechanosensor functions and intracellular protein regulations of 2 2 TRPM7, coupled with other TRPM7 physiological roles in Mg + homeostasis and Ca + signaling implies that the vital and ubiquitous TRPM7 could have crucial roles in cell growth and differentiation, as well as, stress and homeostatic adjustments of membrane 2 tension. TRPM7 is suggested to regulate cell cycle by channel Ca + conductance (Hanano et aI., 2004). TRPM7 over-expression appears to disrupt a cellular mechanism or pathway 98 2 involving Ca +. The rounding, clumping, swelling and detaching effects ofTRPM7 over- expression could be from cells being arrested in the cell cycle. 2 2 TRPM7 may regulate cell volume, intercellular Mg + and Ca +, the cytoskeleton and membrane tension adjustments in the dividing cell. The largest increase in [Mg2+]i occurs during cell proliferation, probably in response to increases in nucleotides and cell volume 2 (Wolf et aI., 2003). The only known ubiquitous Mg + channel is TRPM7 (Nadler et aI., 200 I; Schmitz et aI., 2004). TRPM1, a protein similar to TRPM7 is associated with suppression of melanoma metastasis (Duncan et aI., 1998; Xu et aI., 2001). Tumor suppressors are frequently involved with inhibition of cell proliferation or apoptosis. Endocrine and paracrine factors can initiate cell proliferation or apoptosis often by activating intracellular tyrosine kinases, for example, growth hormones, insulin and insulin-like growth factors. TRPM7 has three tyrosine kinase phosphorylation sites. Two are in regions likely involved in TRPM7 function, the coiled-coil region and the kinase region. In contrast to TRPM7, TRPM6 has no tyrosine kinase phosphorylation sites. Future research will need to examine TRPM7 roles in cell growth, the cytoskeleton, as well as, other physiological events that would use an osmo-mechanosensitive channel, conduct divalent cations, bind proteins involved in cell morphology, aggregation and adhesion and likely phosphoregulate these proteins. 5.3. EXTRACELLULAR GLUTAMATE EFFECTS TRPM7 CURRENTS At negative potentials, TRPM7 carries a divalent cation specific influx, and at positive potentials, primarily a monovalent cation efflux (Nadler et aI., 200 I). However, at low positive potentials, there is a slight divalent cation specific influx inward, which 99 dissipates with increased size ofthe positive voltage. The combination ofthe monovalent cation efflux and the divalent cation influx, produce a distinctive shape to TRPM7 currents at positive potentials. The divalent cation specificity is thought to be by a divalent permeation block of a series of divalent cations in the pore (Hille, 2001; Kerschbaum et aI., 2003). When a divalent cation is in the pore region, monovalent cations'cannot be conducted. TRPM7 permeation block appears to be unidirectional, because it is not reflective of intracellular divalent cation concentrations and dissipates away at positive potentials. The cations are attracted to enter the channel by the negative charge within the cell at negative potentials. At positive potentials, cations are repulsed from the cell. However, negatively charged residues near and in the ion pore region could counter the low positive potentials. It appears that extracellular solution made of Na glutamate diminishes the ability ofthese pore negatively charged residues to attract divalent cations. Replacing the extracellular NaCI with Na-glutamate decreases the slight divalent cation inward permeation block at low positive potentials, but does not effect the divalent cation permeation at negative potentials. Therefore, Na glutamate allows the monovalent cation carried efflux to be larger at lower positive potentials (figure 4.3.1). The disruption of the divalent cation permeation block by Na-glutamate is due to alteration of divalent cation permeation at low positive potentials, and not by changes in channel open probability or chelation of a specific divalent cation. Extracellular Na 2 glutamate effects on TRPM7-currents occur in solutions without extracellular Ca + or 2 Mg +, and do not occur in extracellular solutions where both are excluded (figure 4.3.2). 100 The increase of TRPM7-carried outward currents by Na-glutamate is independent and occurs simultaneous to hypertonic effects. This is likely due to Na-glutamate diminishing the attraction ofdivalent cations to the channel at low positive potentials, while the effect of hypertonic solution is due to closing of the channel (4.3.3). Future research with site- directed mutagenesis of suspected negatively charged residues would bring a greater clarity on how the divalent cations are attracted to the channel at low positive potentials and how Na glutamate can disrupt this ability. 5.4. SUMMARY TRPM7 and TRPM617 channel activity is sensitive to osmolarity, independent of the f-actin cytoskeleton, pH and cAMP. The mechanism of the osmosensitivity may be 2 2 directly by cell-volume changes in Mg + or Mg·ATP concentrations. Mg +, Mg·ATP and a-kinase activation influence the osmotic effects on channel activity. Single-channel patch-clamp research will need to performed in the future to see if the channels are directly responsive to pressure and lipid deformations (mechanosensitive). Possibly, unknown agonists or inhibitory factors or other mechanisms of protein regulation are involved in TRPM7 osmosensitivity. TRPM7 and TRPM617 over-expressing cells become round, swell, clump, detach, but remain viable. The over-expression of the channel and subsequent over-loading the 2 2 2 cytosol with Mg + or Ca + does not appear to be involved. Actually, removing Ca + from the incubation media mimics and increases the kinetics of TRPM7 over-expression effects on cells. TRPM7 and TRPM6 a-kinase is involved in TRPM7 and TRPM617 over-expression induced effects on cells, but it is not necessarily by phosphorylation of 101 proteins. The results suggest the over-expression effects on cells are from binding and thereby, removing proteins involved in cell morphology, adhesion and aggregation. However, structural changes in the over-all protein could be a factor. The only known protein to associate with TRPM7 a-kinase is annexin I, but it is not involved in these effects. Future research with over-expressing the a-kinase with plasma membrane region to bring it near the membrane, but not have any channel activity would clarify the a- kinase involvement in the TRPM7 over-expression induced changes in cell morphology, adhesion and aggregation. TRPM7 channel and kinase most likely work in conjunction with each other. This suggests TRPM7 could be a mediator between membrane strain, the membrane and the proteins involved in cell size, shape, adhesion and aggregation, such as cytoskeletal proteins, annexins and phospholipases that would alter membrane strain, as well as, adjusting intracellular divalent cations. In such a role, TRPM7 could be involved in cell growth and differentiation. In these conditions, cells change size, membranes are 2 deformed and there is a need for adjusting Mg + levels. At positive potentials, TRPM7 channel conductance and divalent permeation is disrupted by extracellular sodium glutamate. This phenomenon might be due to glutamate interfering or competing with pore amino acids attraction of divalent cations. Future research could examine this possibility with site-directed mutagenesis ofthe pore region. 102 REFERENCES Aarts, M., Iihara, K., Wei, W.-L., Xiong, Z.-G., Arundine, M., Cerwinski, W., MacDonald, J. F., and Tymianski, M. (2003). A key role for TRPM7 channels in anoxic neuronal death. Cell, 115: 863-77. Aita, V.M., Liu, J., Knowles, lA., Terwilliger, J.D., Baltazar. R., Grunn. A., Loth. J.E., Kanyas, K., Lerer, R, Endicott, J., Wang, Z., Penchaszadeh, G., Gilliam, T.C. and Baron, M. (1999) A comprehensive linkage analysis of chromosome 21 q22 supports prior evidence for a putative bipolar affective disorder locus. Am J Hum Genet., 64:210-7. . Arniges, M., Vazquez, E., Fernandez-Fernandez, J.M. and Valverde, M.A. (2004) Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J Bioi Chem., 279: 54062-8. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z, Miller, W. and Lipman DJ. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402. Bang, H., Kim, Y. and Kim, D. (2000) TREK-2, a new member ofthe mechanosensitive tandem-pore K+ channel family.J Bioi Chern., 275:17412-9. Batiza, A.F., Schulz, T., and Masson, P.H. (1996) Yeast respond to hypotonic shock with a calcium pulse. 1. Bioi. Chem., 271: 23357-62. Baumgarten, C.M. and Feher, JJ. (2001) Osmosis and regulation of cell volume. In N. Sperelakis (Ed.), Cell physiology sourcebook: A molecular approach, 3rd Ed. (pp. 167 178). San Diego: Academic Press. Benet, L.Z., Kroetz, D.L., and Sheiner, L.R (1996) Pharmacokinetics: The dynamics of drug absorption, distribution and elimination. In Hardman, J.G., Limbird, L.E., Molinoff, P.R, Ruddon, R.W. and Gilman, A.G. (Eds.) Goodman & Gilman's The pharmacological basis oftherapeutics, 9th ed (pp 1-26) New York: McGraw-Hill. Brewster, J.L. and Gustin, M.C. (1994) An osmosensing signal transduction pathway in yeast. Science, 259: 1760-3. Bourque, C.W. and Oliet, S.H. (1997) Osmoreceptors in the central nervous system. Annu Rev Physiol., 59: 601-19. 103 Bucher P., Bairoch A. (1994) A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. In Altman, R., Brutlag, D., Karp, P., Lathrop, R., Searls, D., (Eds.) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology (pp53-61) Menlo Park, CA: AAAI Press, Inc. Card, C. (2000). HEK 293 cells: characteristics and culture. Art to Science, 19: 1-2. Coudray, c., Feillet-Coudray, C., Grizard, D., Tressol, lC., Gueux, E. and Rayssiguier, Y. (2002) Fractional intestinal absorption of magnesium is directly proportional to dietary magnesium intake in rats. 1. Nutr. 132: 2043-2047. - Chubanov, V., Waldegger, S., Mederos y Schnitzler, M., Vitzthum, H., Sassen, M.C., Seyberth, H.W., Konrad, M. and Gudermann, T. (2004). Disruption of TRPM6 TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia. PNAS, 101: 2894-2899. Colbert, H.A., Smith, T.L. and Bargmann, C.L. (1997) OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. 1. Neurosci., 17: 8259-69. Dedman, J.R. & Kaetzel, M.A. (2001). Calcium as an intracellular second messenger: Mediation by calcium-binding proteins. In N. Sperelakis (Ed.), Cell physiology sourcebook: A molecular approach, 3rd Ed. (pp. 167-178). San Diego: Academic Press. de la Roche, M.A., and Cote, G. P. (2001) Regulation of Dictyostelium myosin I and 11. Biochirn Biophys Acta.,1525:245-61. de Nadal, E., Alepuz, P.M. and Posas, F. (2002) Dealing with osmostress through MAP kinase activation. EMBO Reports, 3: 735-40. Dorovkov, M.V., and Ryazanov, A.G. (2004) Phosphorylation of annexin I by TRPM7 channel-kinase. J Bioi Chern., 279: 50643-6. Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S. L., Chait, B.T. and MacKinnon, R. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science., 280: 69-77. Drennan D. and Ryazanov A.G. (2004) Alpha-kinases: analysis of the family and comparison with conventional protein kinases. Prog Biophys Mol BioI., 85: 1-32. Duncan, L.M., Deeds, J., Hunter, J., Shao, J., Holmgren, L.M., Woolf, E.A., Tepper, R.I. and Shyjan, A.W. (1998) Down-regulation of the novel gene melastatin correlates with potential for melanoma metastasis. Cancer Res. 58: 1515-20. 104 Earley, S., Waldron, BJ. and Brayden, lE. (2004) Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ Res., 95: 922-9. Fine, K.D., Santa Ana, e.A., Porter, J.L., and Fordtran, lS. (1991) Intestinal absorption ofmagnesium from food and supplements. J Clin Invest., 88: 396--402. Fleig A. and Penner R. (2004) The TRPM ion channel subfamily: molecular, biophysical and functional features. Trends Pharmacol Sci., 25: 633-9. Fujita-Becker, S., Durrwang, U., Erent, M., Clark, RJ., Geeves, M.A. and Manstein, DJ. (2005) Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I Myosin. J BioI Chem., 280: 6064-71. Gerke, V. and Moss, S.E. (2002) Annexins: from structure to function. Physiol Rev., 82: 331-71. Graham, F.L., Smiley, J., Russell, W.e. and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 36:59-74. Greger, J.L., Smith, S.A. & Snedeker, S.M. (1981). Effect of dietary calcium and phosphorus magnesium, manganese and selenium in adult males. Nutr. Res., 1: 315 325. Grimm, C., Kraft, R., Sauerbruch, S., Schultz, G. and Harteneck, e. (2003) Molecular and functional characterization of the melastatin-related cation channel TRPM3. J. Biochem, 278: 21493-501. Grimm, C., Kraft, R., Schultz, G. and Harteneck, C. (2005) Activation of the melastatin related cation channel TRPM3 by D-erythro-sphingosine. Molec. Pharm . 67: 798 805. Gossen M. and Bujard, H. (1992) Tight control ofgene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl AcadSci USA, 89: 5547-51. Gustin, M.e., Albertyn, J., Alexander, M. and Davenport, K. (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev., 62: 1264 1300. Gustin, M. e., Zhou, x.-L., Martinac, B. and Kung, C. (1988) A mechano-sensitive ion channel in the yeast plasma membrane. Science, 242: 762-65. 105 Gwanyanya, A, Amuzescu, B., Zakharov, S.l., Macianskiene, R., Sipido, K.R., Bolotina, V.M., Vereecke, J., and Mubagwa, K. (2004) Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation. J Physiol, 559: 761-76. Hara, Y., Wakamori, M., Ishii, M., Maeno, E., Nishida, M., Yoshida, T., Yamada, H., Shimizu, S., Mori, E., Kudoh, J., Shimizu, N., Kurose, H., Okada, Y, Imoto, K. and Mori, Y. (2002) LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell., 9: 163-73. Hanano, T., Hara, Y., Shi, 1., Morita, H., Umebayashi, C., Mori, E., Sumimoto, H., Ito, Y., Mori, Y., and Inoue, R. (2004) Involvement of TRPM7 in cell growth as a spontaneously activated Ca2+ entry pathway in human retinoblastoma cells. J Pharmacol Sci., 95: 403-19. Harteneck, e., Plant, T.D., and Schultz, G. (2000) From worm to man: three subfamilies ofTRP channels. Trends Neurosci., 23: 156-166. Hazama, A. and Okada, Y. (1988) Ca+2 sensitivity of volume-regulatory K+ and cr channels in cultured human epithelial cells. 1. Physiology, 402: 687-702. Hazama, A. and Okada, Y. (1990) Involvement of Ca+2 -induced Ca+2 release in the volume regulation ofhuman epithelial cells exposed to a hypotonic medium. Biochem. & Biophys. Res. Comm., 167: 287-293. Hermosura, M.e., Monteilh-Zoller, M.K., Scharenberg, AM., Penner, R., and Fleig, A (2002). Dissociation of the store-operated calcium current I(CRAC) and the Mg nucleotide-regulated metal ion current MagNuM. 1. Physiol., 539: 445-458. Hille, B. (2001) Ion Channels ofExcitable Membranes, 3rd Ed. Sunderland, MA: Sinauer Associates. Hohmann, S. (2002) Osmotic stress signaling and osmoadaptation III yeasts. Microbiology & Molecular Biology Reviews, 66: 300-72. Huang, C.-L. (2004) The transient receptor potential superfamily of ion channels. Am. 1. ofNephrology, 15: 1690-9. Iqbal, J. and Zaidi, M. (2005) Molecular regulation of mechanotransduction. Biochem Biophys Res Commun., 328:751-5. Jewett, D.L. and Rayner, M.D. (1984) Basic Concepts of Neuronal Function. Boston: Little Brown. 106 Jiang, X., Newell, E.W. and Schlichter, L.c. (2003) Regulation of a TRPM7-like current in rat brain microglia. 1. Bioi. Chem., 278: 42867-76. John, C.D., Christian, H.C., Morris, J.F., Flower, RJ., Solito, E. and Buckingham, J.C. (2004) Annexin 1 and the regulation ofendocrine function. Trends Endocrinol Metab. 15:103-9. Kanzaki, M., Nagasawa, M., Kojima, 1., Sato, c., Naruse, K., Sokabe, M. and Iida, H. (1999) Molecular identification of a eukaryotic, stretch-activated nonselective cation channel. Science, 285: 882-6. Kerschbaum, H.H., Kozak, J.A., and Cahalan, M.D. (2003). Polyvalent cations as permeant probes ofMIC and TRPM7 pores. Biophysical Journal, 84: 2293-2305. Ko, B.C., Lam, AX., Kapus, A., Fan, L., Chung, S.K. and Chung, S.S. (2002). Fyn and p38 signaling are both required for maximal hypertonic activation of the osmotic response element-binding protein/tonicity-responsive enhancer-binding protein (OREBP/TonEBP). J Bioi Chem., 277: 46085-92. Kozak, J.A., Kerschbaum, H.H., and Cahalan M.D. (2002) Distinct properties of CRAC and MIC channels in RBL cells. J Gen Physiol. 120: 221-35. Kozak, lA. and Cahalan, M.D. (2003) MIC channels are inhibited by internal divalent cations but not ATP. Biophys 1., 84: 922-7. Kozak, lA., Kerschbaum, H.H., and Cahalan M.D. (2004) Regulation of MIC and TRPM7 channels by internal polyvalent cations (Poster).48th Biophysical Society Meeting, Baltimore, MD Kraft, R., Grimm, c., Grosse, K., Hoffmann, A., Sauerbruch, S., Kettenmann, H., Schultz, G. and Harteneck, C. (2004) Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia. Am J Physiol Cell Physiol, 286: C129-37. Ktiltz, D. and Burg, M. (1998) Evolution of osmotic stress signaling via MAP kinase cascades. 1. Exp. Bioi., 201: 3015-3021. Langeslang, M., van Leeuwen, F., and Jalink, K. (2004) N 1E-115 over-expressing 2 th TRPM7 display increased MIC and altered Ca + homeostasis (Poster).48 Biophysical Society Meeting, Baltimore, MD Launay, P., Fleig, A., Perraud, A.L., Scharenberg, A.M., Penner, R. and Kinet, J.P. (2002) TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell. 109: 397-407. 107 Launay, P., Cheng, H., Srivatsan, S., Penner, R., Fleig, A. and Kinet, J.P. (2004) TRPM4 regulates calcium oscillations after T cell activation. Science, 306: 1374-7. Laevsky, G. and Knecht, D.A. (2003) Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments. J Cell Sci., 116: 3761-70. Lee, N., Chen, J., Sun, L., Wu, S., Gray, K.R., Rich, A., Huang, M., Lin, J.H., Feder, J.N., Janovitz, E.B., Levesque, P.C. and Blanar, M.A. (2003) Expression and characterization of human transient receptor potential melastatin 3 (hTRPM3). J BioI Chern., 278: 20890-7. Liedtke, W., Tobin, D.M., Bargmann, c.1. and Friedman, J.M. (2003) Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and mechanical stimuli in Caenorhabditis elegans. PNAS, 100: 14531-6. Liedtke, W., Choe, Y., Marti-Renom, M. A., Bell, A.M., Denis, C.S., Sali, A., Hudspeth, AJ., Friedman, J.M. and Heller, S. (2000) Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell, 103: 525-35. Liu, D. and Liman, E.R. (2003) Intracellular Ca2+ and the phospholipid PIP2 regulate the taste transduction ion channel TRPM5. Proc Natl Acad Sci USA, 100:15160-5. Lordish, H., Beck, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. and Darnell, J. (2000) Cell motility and shape I: microfilaments. Molecular cell biology 4th Ed. New York: Freeman & Co. Lupas, A., Van Dyke, M. and Stock, J. (1991) Predicting coiled coils from protein sequences. Science, 252:1162-1164. Maingret, F., Patel, AJ., Lesage, F., Lazdunski, M. and Honore E. (1999) Mechano- or acid stimulation, two interactive modes of activation of the TREK-l potassium channel. J BioI Chern., 274: 26691-6. Maingret, F., Fosset, M., Lesage, F., Lazdunski, M. and Honore, E. (1999) TRAAK is a mammalian neuronal mechano-gated K+ channel. J BioI Chern. 274: 1381-7. Maeda, T., Wurgler, S.M. and Saito, H. (1994) A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature, 369: 242-45. Maguire, M.E. (1982) Magnesium regulation of the beta-receptor-adenylate cyclase complex. II. Sc3+ as a Mg2+ antagonist. Mol. Pharm. 22: 274-280. 108 Maroto, R., Raso, A., Wood, T.G., Kurosky, A., Martinac, B. and Hamill, O.P. (2005) TRPC1 forms the stretch-activated cation channel in vertebrate cells. Nat Cell BioI. 7: 179-85. Matthews, G., Neher, E. and Penner R. (1989) Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells. J Physiol, 418: 131-44. Meij, I.C., van den Heuvel L.P. and Knoers. N.V. (2002) Genetic disorders ofmagnesium homeostasis. Biometals, 15: 297-307. Mizuno, A., Matsumoto, N., Imai, M., and Suzuki, M. (2003) Impaired osmotic sensation in mice lacking TRPV4. Am J Physiol Cell Physiol., 285: C96-101. Monteilh-Zoller, M.K., Hermosura, M.C., Nadler, MJ., Scharenberg, A.M., Penner, R. and Fleig, A (2003) TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. 1. Gen. Physiol., 121: 49-60. Montell, C., Bimbaumer, L., Flockerzi, V., Bindels, RJ., Bruford, E.A, Caterina, MJ., Clapham, D.E., Harteneck, c., Heller, S., Julius, D., Kojima, 1., Mori, Y, Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Schimizu, N. and Zhu, M.X. (2002) A unified nomenclature for the superfamily of TRP cation channels. Molecular Cell, 9: 229-231. Murakami, M., Xu, F., Miyoshi, 1., Sato, E., Ono, K. and lijima, T. (2003) Identification and characterization of the murine TRPM4 channel. Biochem Biophys Res Commun. 307: 522-8. Nadler, MJ.S., Hermosura, M.C., Inabe, K., Perraud, A-L., Zhu, Q., Stokes, A. J., Kurosaki, T., Kinet, J.-P., Penner, R., Scharenberg, A.M. and Fleig, A. (2001) LTRPC7 is a Mg'ATP-regulated divalent cation channel required for cell viability. Nature, 411: 590-595. Nagamine, K., Kudoh, J., Minoshima, S., Kawasaki, K., Asakawa, S., Ito, F. and Shimizu, N. (1998) Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain. Genomics, 54: 124-31. Nemeth, E.F. (2001). Regulation of cellular functions by extracellular calcium. In N. Sperelakis (Ed.), Cell physiology sourcebook: A molecular approach, 3rd Ed. (pp. 167 178). San Diego: Academic Press. 2 Oiki, S., Kubo, M. and Okada, Y. (1995) Mg + and ATP-dependence ofvolume-sensitive cr channels in human epithelial cells. Japanese Journal ofPhysiology, 44: S77-S79. 109 Okada, Y., Maeno, E., Shimizu, T., Dezaki, K., Wang, J., and Morishima, S. (2001) Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). Journal ofPhysiology, 532: 3-16. Paidhungat, M. and Garrett, S. (1997) A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdcl(Ts) growth defect. Mol Cell BioI. 17: 6339-47. Patel AJ. and Honore, E. (2001) Properties and modulation of mammalian 2P domain K+ channels. Trends Neurosci. 24: 339-46. Penner, R. (1995) A practical guide to patch clamping. In Sakmann, B. and Neher, E. (Eds.) Single-channel recording 2nd Ed. New York: Plenum Press. Perraud, AL., Fleig, A, Dunn, C.A., Bagley, L.A, Launay, P., Schmitz, C., Stokes, AJ., Zhu, Q., Bessman, MJ., Penner, R., Kinet, lP. and Scharenberg A.M.ADP-ribose gating ofthe calcium-permeable LTRPC2 channel revealed by Nudix motif homology. Nature, 411: 595-9. Peier, A.M., Moqrich, A., Hergarden, A.C., Reeve, AJ., Andersson, D.A., Story, G.M., Earley, TJ., Dragoni, 1., McIntyre, P., Bevan, S. and Patapoutian, A. (2002) A TRP channel that senses cold stimuli and menthol. Cell, 108: 705-15. Perez, C. A, Huang, L., Rong, M., Kozak, lA., Preuss, A.K., Zhang, H., Max, M. and Margolskee, R.F. (2002) A transient receptor potential channel expressed in taste receptor cells. Nat Neurosci., 5: 1169-76. Prawitt, D., Monteilh-Zoller, M.K., Brixel, L., Spangenberg, c., Zabel, B., Fleig, A. and Penner, R. (2003) TRPM5 is a transient Ca2+-activated cation channel responding to rapid changes in [Ca2+]i. Proc Natl AcadSci USA. 100:15166-71. Rosette, C. and Karin, M. (1996) Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science, 274: 1194-97. Roviezzo, F., Getting, SJ., Paul-Clark, MJ., Yona, S., Gavins, F.N., Perretti, M., Hannon, R., Croxtall, J.D., Buckingham, lC. and Flower, RJ. (2002) The annexin-I knockout mouse: what it tells us about the inflammatory response. J Physiol Pharmacol., 53: 541-53. Runnels, L. W., Yue, L. and Clapham, D.E. (2001) TRP-PLlK, a bifunctional protein with kinase and ion channel activities. Science, 291: 1043-1047. Runnels, L. W., Yue, L. and Clapham, D. E. (2002) The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat. Cell BioI., 4: 329-36. 110 Ryazanov, A.G., Pavur, K.S. and Dorovkov, M.V. (1999) Alpha-kinases: a new class of protein kinases with a novel catalytic domain. Curro BioI., 9: R43-R45. Ryazanov A.G. (2001) Elongation factor-2 kinase and its newly discovered relatives. FEBS Lett., 5]4: 26-9. Ryazanova, L.V., Dorovkov, M.V., Ansari, A. and Ryazanov, A.G. (2004) Characterization of the protein kinase activity of TRPM7/ChaKl, a protein kinase fused to the transient receptor potential ion channel. 1. Bio!. Chern., 279: 3708-16. Sardini, A., Arney, J.S., Weylandt, K.H., Nobles, M., Valverde, M.A., and Higgins, C.F. (2003) Cell volume regulation and swelling-activated chloride channels. Biochirn Biophys Acta. 1618:153-62. 2 Satoh, J. and Romero, M.F. (2002) Mg + transport in the kidney. BioMetals, 15: 285-295. Schlingmann, K.P., Weber, S., Peters, M., Niemann Nejsum, L., Vitzthum, H., Klingel, K., Kratz, M., Haddad, E., Ristoff, E., Dinour, D., Syrrou, M., Nielsen, S., Sassen, M., Waldegger, S., Seyberth, H.W., and Konrad, M. (2002) Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet.,31:166-70. Schmitz, e., Perraud, A.L., Johnson, e.O., Inabe, K., Smith, M.K., Penner, R., Kurosaki, T., Fleig, A., Scharenberg, A.M. (2003) Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell, 114:191-200. Shaw, G., Morse, S., Ararat, M. and Graham, F.L. (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin ofHEK 293 cells. FASEB 1., 16: 869-71. Sheikh-Hamad, D., and Gustin, M.e. (2004) MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. Am J Physiol Renal Physiol., 287: Fll02-1 O. Simon, D.B., Lu, Y., Choate, K.A., Velazquez, H., Al-Sabban, E., Praga, M., Casari, G., Bettineli, A., Colussi, G., Rodriguez-Soriano, 1., McCredie, D., Milford, D., Sanjad, S. and Lifton, R.P. (1999) Paracellin-l, a renal tight junction protein required for 2 paracellular Mg + resorption. Science, 285: 103-6. 111 Solito, E., Mulla, A., Morris, J. F., Christian, H.C., Flower, RJ. and Buckingham, J.C. (2003) Dexamethasone induces rapid serine-phosphorylation and membrane translocation of annexin 1 in a human folliculostellate cell line via a novel nongenomic mechanism involving the glucocorticoid receptor, protein kinase C, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase. Endocrinology. 144: 1164-74. Solomon, A., Bandhakavi, S., Jabbar, S., Shah, R., Beitel, GJ. and Morimoto, R.I. (2004) Caenorhabditis elegans OSR-l regulates behavioral and physiological responses to hyperosmotic environments. Genetics, 167:161-70. Strotmann, R., Harteneck, c., Nunnenmacher, K., Schultz, G. and Plant, T.D. (2000) OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat. Cell Bioi., 2: 695-702. Suzuki, M., Mizuno, A., Kodaira, K. and Imai, M. (2003) Impaired pressure sensation in mice lacking TRPV4. J BioI Chem, 278: 22664-8. Tian, W., Salanova, M., Xu, H., Lindsley, J.N., Oyama, T.T., Anderson, S., Bachmann, S. and Cohen, D.M. (2004) Renal expression of osmotically responsive cation channel TRPV4 is restricted to water-impermeant nephron segments. Am J Physiol Renal Physiol., 287: F17-24. Takezawa, R., Schmitz, C., Demeuse, P., Scharenberg, A.M., Penner, R., and Fleig, A. (2004) Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain. Proc Natl AcadSci USA, 101:6009-14. Thompson, J.D., Higgins, D.G. and Gibson, T.1. "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice." httrlJ/bimass.dcrt.nih.g~)V/cIustalw/clustalw.html UN WHO and FAO (2002) Magnesium. FAO/WHO expert consultation on human vitamin and mineral requirements. b.1!p:/IJ}.':.w\\.:'Jao.org/docll;nents van der Wijk,T., Tomassen, S.F., de Jonge, H.R. and Tilly, B.c. (2000) Signalling mechanisms involved in volume regulation of intestinal epithelial cells. Cell Physiol Biochem., ]0: 289-96. Voets, 1., Nilius, B., Hoefs, S., van der Kemp, A.W.C.M., Droogmans, G., Bindels, R. J. M & Hoenderop, J. G. J. (2004) TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. JBC, 279: 19-25. 112 Vriens J, Watanabe H, Janssens A, Droogmans G, Voets T, Nilius B. (2004) Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4. Proc Natl Acad Sci US A,lOl: 396-401. Walder, R.Y., Shalev, H., Brennan, T.M., Carmi, R., ElbedoUf, K., Scott, D.A., Hanauer, A., Mark, A.L., Patil, S., Stone, E.M. and Sheffield, V.C. (1997) Familial hypomagnesemia maps to chromosome 9q, not to the X chromosome: genetic linkage mapping and analysis of a balanced translocation breakpoint. Hum Mol Genet., 6:1491-7. Walder, R. Y., Landau, D., Meyer, P., Shalev, H., Tsolia, M., Borochowitz, Z., Boettger, M.B., Beck,"G.E., Englehardt, R.K., Carmi, R., and Sheffield, V.C. (2002) Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet., 31:171-4. Watanabe, H., Vriens, J., Prenen, J., Droogmans, G., Voets, T. and Nilius, B. (2003) Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature, 424: 434-8. Wehage, E., Eisfeld, J., Heiner, 1., Jungling, E., Zitt, C. and Luckhoff, A. (2002) Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose. J Bioi Chern., 277: 23]50-6. Wolf, F.1., Torsello, A., Fasanella, S. and Cittadini, A. (2003) Cell physiology of magnesium. Molecular Aspects ofMedicine, 24: 11-26. Wood, J.M. (1999) Osmosensing by bacteria: signals and membrane-based sensors. Microbiology & Molecular Biology Reviews, 66: 230-262. Xu, X.Z., Moebius, F., Gill, D.L., and Montell, C. (2001) Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform. Proc Natl Acad Sci USA, 98: 10692-7. Xu, H., Zhao, H., Tian, W., Yoshida, K., Roullet, J.B. and Cohen, D.M. (2003). Regulation of a transient receptor potential (TRP) channel by tyrosine phosphorylation. SRC family kinase-dependent tyrosine phosphorylation of TRPV4 on TYR-253 mediates its response to hypotonic stress. J Bioi Chern. 278: 11520-7. Yamaguchi, H., Matsushita, M., Nairn, A.C. and Kuriyan, 1. (2001) Crystal structure of the atypical protein kinase domain ofa TRP channel with phosphotransferase activity. Mol. Cell, 7:1047-57. Yoon, 1.S., Li, P.P., Siu, K.P., Kennedy, 1.L., Macciardi, F., Cooke, R.G., Parikh, S.V. and Warsh, J.J. (2001) Altered TRPC7 gene expression in bipolar-I disorder. Bioi Psychiatfy, 50: 620-6. 113 Zhang, L. and Barritt, G.J. (2004) Evidence that TRPM8 is an androgen-dependent Ca2+ channel required for the survival ofprostate cancer cells. Cancer Res. 64: 8365-73. Zhou, Y., Morais-Cabral, J.H., Kaufman, A. and MacKinnon, R. (2001) Chemistry ofion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature, 414: 43-8. APPENDIX I. TRPM AMINO ACID SEQUENCE AUGNMENT TRPM3 mpep- -wgtvyflgi aqvfslf. f wwn; eg- ..·mngadaprpl,[lli Jrklchaaflpsvrllkaq',ls Wi erafykrecvhi lpStkdphr TRPM6 ------mkeqpvlerlq qk~wikgvfdkre~ ~ .knph TRPM7 ------m~qk~wiesrltkrecvyiip ~kdphr TRPM4 ------mvvpeke-q----swipkifkkk(cttfiVd~[dpgg TRPM2 -mepsalrkagseqeegfeglprrvtdlgmvsnlrr~nsslfk~wrlqcpfgnnd----kqe ls'wipenikkkecvyfv e~skagk TRPM8 ------m' fraarl-m--rnrrndtld,trtly ~asr~tdlsys esdlvnfiqanfkkrecvffi TRPM3 C---C------ncgrL ;gqh'.gl J21 vlgnel TRPM3 kkreeevdidlddpei nhfpfpfhelmv-wavlmkr,gkma -ffwghgeeamaka l.ack -~ckamaheasendmvt1dl '"1'"' " ..... ~ TRPMl kkkeeeididvddpavsrfqypfhelmv-wavlmkrqkmtlwgcgee ~l ..'ack ~ykamahessesdlvddi gdldnn kd TRPM6 rkk,keQll~ ~dpestg-flypyndllv-wavlmkrqkmamffwqhgeeatvk~vi~~lyramaheakeshmvddajeel~n TRPM7 kkkr kdeivdiddpetkrfpyplnelliwaclmkrqvmarflwghgee makaL-ack ~lyrsmayeakqsdlv'QQ. ee lk TRPM4 pwsdll1wall1nraqmamyfwemg~navssalgacl1lrvmarlepdaeeaarrkdlafkfegmgvdlfgecyrsse TRPMS pwrdlflwavlqnrhematyfwamgqegvaaalaackilkemshleteaeaaratre-a-kyerlaldlfsecysnse TRPM2 pi rdl1iwalvqnrrelagiiwaqsqdciaaalacskilkelskeeedtdsseemlalaeeyehraigvftecyrkde TRPM8 plqalfiwailqnkkelskviweqtrgctlaalgaskl1ktlakvkndlnaageseelaneyetravelftecyssde TRPM3 gqlavelldqsykqdeqlamklltyel~nw_na Glqlavaakhrdfiahtcsqmlltdmwmgrlrmrkn Iglkvilgil1ppsilsle---~ TRPMl gqlalelldqsykhdeqiamklltyel~nw n ~lklavaakhrdfiahtc5qmlltdmwmgrlrmrknpglkvimgillpptllfle---fr-t TRPM6 gqlaldllekafkqnermamtlltyelrpw n~lklavsgglrpfvshtctqmlltdmwmgrlk~rknlwlkiii~ill_pptlItle---fk-s TRPM7 gqlavelleqsfrqdetmamklltyelk'nw 11 .Qlklavssrl rpfvahtctqmllsdmwmgrlnmrlsn Iwykvi l~ll Yoga ILl Le- --yk~t TRPM4 vraarlllrrcpl------wgda+clqlamqadaraffaqdgvqslltqkwwgdmasttpiwalvlaffc-ppliytrlitfrks TRPMS arafallvrrnrc------wskttclhlateadakaffahdgvqafltriwwgdmaagtpilrllgaflc-palvytnlitf--s TRPM2 eraqklltrvsea------wgkttclqlaleakdmkfvshggiqafltkvwwgqlsvdnglwrvtlcmlafpl11tg-lisfrek TRPM8 dlaeqllvyscea------wggsnclelaveatdqhfiaqpgvqnflskqwygeisrdtknwkiilclfiiplvgcgf-vsfrkk TRPM3 kddmp¥msqaqeihlqekeaeepekptkekeeedmeltamlgrnngessrkkdeeevq khrlljQlgrk;ye TRPMl yddfsyqts------kened-gkekeeentdanadag------srkgdeenehkkgrl plgkice TRPM6 kaemshvpqsqd-fqfmwyysdqnassskesasvkeydlerghdekldenqh--fglesghqhl-pwtrkvye n TRPM7 kaemshipqsqdahqmtmgg ennfgni'~ipmevfkevrildsn-egkneme--iqmkskkl-pitrkfya TRPM4 eeeptreelefdmdsvingegpvgtadpaektplgvprqsgrpgccggrcggrrclrrwfh TRPMS eeaplrtgledlqdldsldteksplyglqsrveelve---apraqgdrgpravflltrwrk TRPM2 ------rlqdvgtpaarara TRPM8 ------pvdkhkkllwyyva TRPM3 p---~tqewiv15yl-- ~~~,---~ ~I ~ ~ TRPMl na'" I ,:kfwf : i 1foy'.' . rmd -. ew I, 'L_' TRPM6 ysapivkwfy-lmaylaflmlftytvlvemqp p-nsvqek£] ..y--f na:e."reic TRPM7 yhapil/kfwfn ~aUf1mlytfvvlvqmeq p---svqewiviayi--ftyaiekvreifl1\ a k'.n· gklk'Jwfd TRPM4 wgapvtifmgnvvsyllflllfsrvllvdfqp ppg-sl-e- ll-yfwaftllceelrqglsg ggslasggpgpghaslsqrlrlylad TRPMS wgapvtvflgnvvmyfaflflftyvllvdfrp pqgpsgpev11--yfwvftlvleeirqgfft edthlv-kkftlyvgd TRPM2 ftapvvvfhlnilsyfaflclfayvlmvdfqp p---swcec i--ylwlfslvceemrqlfy- pdecglmkkaalyfsd TRPM8 ftspfvvfswnvvfyiaflllfayvllmdfhs php-p--el l--yslvfvlfcdevrqwyvn nksslys----n-yftd TRPM3 ywn" d~ j a:: Lt"drJll) Lr ~q- n - n ---dqpfrsdgrvi YCVnl; ywyi r1lld J.f.g\·JJ.!sYJ~vmmi gkmmi dmm~fvi iml vvlm TRPMl yJn. ,,dl yah fmi gailrlq--nn---nqpymgygrvlycvdi ifwyi 0ild 1fg:oky 1gpY'lmmi $mni dm iyfvvimlvvlm TRPM6 ywnl ~tvalglf agfvlrwg------dppf~tagrliycidllfwf5r!ldffavnqhagpyvtmiakmtanmfYiViimaivll TRPM7 yfUI fJ1 i ai i ~ff; gfglrfgakwnfanaydnhv,f. agr .Y£-'n. fwyvrlLdf ~ avnqqagpyvrnmi gkmvanmfyivvimal vl TRPM4 swnqcdlvaltcfllgvgcrltp------glyhlgrtvlcidfmvftvrllhiftvnkqlgpkivivskmmkdvffflfflgvwlv TRPMS nwnkcdmvaiflfivgvtcrmlp------safeagrtvlamdfmvftlrlihifaihkqlgpkiivvermmkdvffflfflsvwlv TRPM2 fwnkldvgaillfvagltcrlip------atlypgrvilsldfilfclrlmhiftisktlgpkiiivkrmmkdvffflfllavwvv TRPM8 lwnvmdtlglfyfiagivfrlhss ------grvifcldyiiftlrlihiftvsrnlgpkiimlqrmlidvffflflfavwmvgv TRPM3 ~fgvarqal1fpneepswklaknifympywmiygevfa-dqid------p-pcgqnenedgkiiqlpp------ckt--- TRPMl fgvarqailhpeekpswklarnifympywnllygevfa-dqidlya-meinp-pcgenlyde~l--pp------cip----- TRPM61 ig~. arka' : ~ppswslardi vfepyWl1\ yge'lyage- idnn------vcssq---- nnn--p--n-SCpp----- TRPM7 .f.g .PIka Ivpheapswtlakdi vfhpywmi fgevyaye- id- -n ---n-vca-nd \1- - n -i ---pqi ----cgp- n -- TRPM4 aygvategllrprdsdfpsilrrvfyrpylqifgqipq-edmdv-alme-hs-ncssep----gfwah-ppgaqagtc-v- r ----- qy TRPMS aygvttqallhphdgrlewlfrrvlyrpylqlfgqipl-deide-arv--n---csthpll-----ledsp-----sc-p-~- - ly TRPM2 sfgvakqail1hnerrvdwlfrgavyhsyltifgqipgy--id--gvnf-npehcs--pn---gtd----pykpk--c-pesdatqqrpaf TRPM8 afgvarqgilrqneqrwrwifr~vlyepylamfgqvp~ d-vd--gttyd-fahctft-- - gnesk--pl-----c-veldehnl-prf TRPM3 gawivpaimacyllvani llvnlliavtruJ. Jifev~ i ~ng'.,wkfgdyql imtfherpvlpppl i ifshmtmifqhlccr-wrkhesdi 0.. TRPMl gawltpalmacyllvanillvnlliav~fevk~i nqvwkfqryqlimtfhdrpvlpppmiil~hlyiiimrlsgrcrkkregdq TRPM6 gsfltpflqavylfvqyi imvnll i affnnvyldmg 1 llOlwkynryryfmtyhekpwlpppl i 11shvglllrrl--cchra-phdq TRPM7 gtwltpflqavylfvqyiimvnlliaffnnvylqvkai~nlvwkyqryhflmayhekpvlpppliilshi-v~---lfccickrrkkdk TRPM4 anwlvvl11vifllvanillvnl1iamf~ytfgkvqgnsdlywkaqryrlirefhsrpalappfivi'hlrlllrql-crrprspqpsJ TRPMS anwlvilllvtfllvtnvllmnlliamf,ytfqvvqgnadmfwkfqrynliveyherpalappfillshlsltlrrvfkkeaehkr TRPM2 pewltvll1clyl1ftnill1nlliamf~qqvqehldqiwkfqrhdlieeyhgrpaapppfil1shlqlfikrvvlktpakrh TRPM8 pewitiplvciymlstnillvnllvamfgytvgtvqenndqvwkfqryflvqeycsrlnipfpfivfayfymvvkkcfkccckeknmessvcc TRPM3 pderdyglklf-lddelkkvhdfeeqcieeyf------rekddrfn nd-erirvI 'ervenm ~rleevnereh-smkaslqtvdir TRPMl -eerdrglklfl,deelkrlhefeeqcvqehf------rekedeqq d-er,rvt~ervenm mrleeineretf-mktslq vdlr TRPM6 -eegdvglklybkedlkklhdfeeqcvekyf------hekmedvm; ce-erir.' erv~yfqlkemnek'lsfi -kdsl1 c::ldc;q TRPM7 tc;d-gpkl f1 teedqkklhdfeeqcvemyf------nekddkfh~~e-eri r .fer.egmciqike/gdr\in\n -kr lqsldc.,q TRPM4 palehf--rvylskeaerklltwe~vhke-nfllara--rdkre---~d e--rlkrt~qkvd---lalkqlghirey-eqr-lkvlere TRPMS -ehlerdlpdpl----dqkvvtwetvqken-fl------~kmekrrrd egevlrktahrvd--fia-k------ylgg--lr--eqe TRPM2 -----kqlknklekneeaallsweiylkeny-lqnrqfqqkqrpeqkle------disnkvdamvdldldplkr~g~meq- TRPM8 ------f--knednetlawegvmkeny-lvkintkandtseemrhrfrqldtklndlkgllkeiankik TRPM3 laqledli grmatalerltglerae-;n- -kl rsr tssdctdaayi vrq-, fnsqegnt -fklqesi dpag-ee LIn -I;I.....J TRPMl laqleelsnrmv~enlag:dr gi l--qarsras~-cea-tyllrq5sin5adgY51yryhfngeellfedtslsts TRPM6 vghlqdl-altvdtlkvl avdtlqedeal1a~rkh tCkklph wc;nvicaevlgsmeiagekkyqyysmpssll-r~l TRPM7 ighlqdlsaltvdtlktltaqka5ea~ - - kvhnei~rel~i~khlaqnliddgpvrpc;vwkk----hgvvntlss~l TRPM4 Vq-q------csrvlgwvaeals-rsall------ppggppppdlpgskd- TRPMS --krikclesqinycsvlvssvadvlaqgggpqh----cgegsqlvaadhrrssggldgweq--pgagqpp----s-dt TRPM2 Kinase domain at (-term end TRPM3 TRPMl . ______._ TRPM6 aggrhpprvqrgalleitnskreatnvrndqerqet~siv' g\ pnrgah k,o..,. ' TRPM7 pqgdle-snnpfhcnilmkddkdpqcnifgqdlpavpgrkefnfpeag gaLfg ~ QQelrqrlhgv-ell--kifn TRPM3 CdldRlddsvnilglgepsfstpvpstapsssayatlaptQrQQL 'dfed!ltsmdtrsfssdythlpecqnpwdseppmyhtierskss TRPMl pclvlhgqdksdvqntqltvettniegtisypleetkitryfp----detinacktmksrsfvyslllllvgg------TRPM6 teqdiqtevlvhltgqtpvvsdwasvdepkekhepiahlldgqdkaeqvlptlsctpepmtmssplsqakimqtgggyvnwafsegdet ~ TRPM7 knqklgssstsiphlsspptkffvstpsqpsckshletgtkdqetvcskategdntefgafvghrdsmdlqrfketsnkik TRPM3 rylattpflleeap1vk_D lmfspsrsyyanfgvp-vk' aeyt it ------dcidtrcvnaQ9a~adraafpggLgdk TRPMl ------vnqdnk dvey~' 'tdqqlttewqcqvqkltrsh~tdlpyiveaavqaehk TRPM6 gVfsikkkwqtclp5tcdsdJsrseqhqkqaqd~~15~, tsaqsj ------ecsevgpwlqpnL~winplrryrpfar~h~ TRPM7 ------itsnll!J. ._en lk I 5 lagftdchrtsi pVhskqaekl --s:r ra It-edthev TRPM3 ,ed: cc..b.pereae-lhpssgHneakgrra ;a1 .qegdn"erllsn----..pkieransysaeepsap TRPMl eqfadmqdehhv-aeaisprlprl------sltit--drngmenlls--vkpdqtlgfpslrskslhghprnvk TRPM6 frfhkeeklmki cki knbgc: se;igggaW\kakml.kdrr. kkkknqglqvpi i tvnacsqsdqlnpe-pgens i TRPM7 dskaalipdwlqdrpsnrempse-egtlng------TRPM3 yahtrksfsisdkldrqrnta lrnpfqrsk5skpegrggslsrnrr1Jrt~afqsfeskhn TRPMl siqgkldrsghaJ5vsslvivsgmtaeekkvkkekastetec TRPM6 seeeyc;knwftvc::kfshtgvepyi hqkmktkei gqcalqbdylkq qedl c;,knslwnsrs lnlnrnr., nk~ I g'.'dk: ,as1 TRPM7 ------It pf TRPM6 kspgephhhysaiernnlmrlsqtipftpvqlfageeitvyrlees~plnldksmsswsqrgraamiqvlsreemdgglrk TRPM7 ,kpamd ~ysavernnlmrl t,ql); pftpvpp-rgepvtvyrlee~>"pni lnnc;msswsqlglcaki efbkeemgggl rr TRPM2 ------rlasleeqvaqtaralhwivrtlrasgfsskaaeepdaepggrkkteepgdsyhvnarhlly pncpvtrfpvpnp TRPM6 amrvvc:twseddilkpgqvfivk~flpevvrtwhkifqestvlhlclre;qqqraaqkliytfnqvkpqtipytprflevfliychs TRPM7 aVkvqctwsehdilksghlyiikJflpevvntwssiykedtvlhlclreiqqqraaqkltfafnqmkpksipysprflevfllychs TRPM2 ekvwetefl,ydppfytaerkdaaamdpmgdtlepl~tlqynvvdglrdrr~fhgpytvqaglplnpmgr glrgrgslscfgpnht TRPM6 anqwltiekymtgefrkynnnngdei ptntleelmlafshw yeytrgellvldlqgvgenltdp~vlkpevkqsrgmvfgpanlgeda TRPM7 agqwfaveecmtgefrkynnnngdeiiptntleeimlafshw yeytrgellvldlqgvgenltdpsvikaeekrscdmvfgpanlgeda TRPM2 lypmvtrwrrnedgalcrksikkmlevlvvklplsehwalpgg repgemlprklkrilrqehwpsfenllkcgmevykgymddprntdn TRPM6 irnflakhhcnsccrklklpdlkrndy ~;n -fgl~kiec:aeepparet grnspeddmql TRPM7 iknfrakhhcnsccrklklpdlkrndytpdkiifpqdepsdlnlqpgu ~e~ec;tn"vrl-m-l TRPM2 awietvavc;vhfqdqndvelnrlnsnlhacdsgasirwqvvdrriplyanhktllqkaaaefgahy M') APPENDIXII. TRPM6 & TRPM7 SEQUENCE ALIGNMENT WITH SIGNATURE MOTIFS TRPM6 mkeqpvlerlqsqkswikgvfdkrec . i jgknphrc gJcqvcqn11rcycgrligdhagidyswtisaakgke------ TRPM7 ------msqkswi estltkrecvyi ipsskdph r£ ~ pgcg i cgg ..~ftas1amkysdvk19dhfnqai G-protein sig C-lectin sig EGF-like 1 TRPM6 seqwsvekhttksptdtfgtinfqdgehthhakyirtsydtkldhllhlmlkewkmelpklvilvhggiqof~pskfkei TRPM7 ee-wsvekhteqsptdaygvinfqggshsyrakyvrl~ydlkpevilqll1kewqmelpklvisvhggmqkfelhprikql Hemeh202sig TRPM6 fsqglvkaaet~gawiitegin~gvskhvgdalkshsshslrkiwtvgippwgvienqrdligkdvvclyqtldnplsklttlnsmhsh TRPM7 ~. I kaa. ga~: z.g' n ~akh.gdakehasrssrki cti gi apwgvi enrndlvgrdvvapyqtllnplsklnvlnnlhsh PK ATP binding WD Repeat TRPM6 fi 1sddgtvgkygnemkl rrnlekylslqki hcrsrqgvpvvgh,eggpn. i, Iwe,vkdkd- -pvvvcegtgraadl1afthkhl-a TRPM7 filvddgtvgkygaevrlrrelektinqqriharigqgvpvvalifeggpnviltVleylqesppvpvvvcegtgraja~~a~ihkqtlap pancrRiboNuclease TRPM6 deg-ml rpqvke-ei i,cm, gn tnt I kqskhl fqi lmecmvhrdci t i fdad-seeqqdldlai 1tallkgtUi ~seqlnlam TRPM7 eeggnlp-daaepdii~tikktfnfgqnealhlfqtlmecmkrkelitvfhi-gsdehqdidvailtallkgtn@~fdqliltl G-protein sig TRPM6 awdrvdi akkhi 1iyeq-hwkpdaleqamsdalvmdrvdfk·, lleyg. n"hrf ,___"prleelyntkqgptn-tllhhlvqdvkq-ht TRPM7 aWdrvdiaknhvfvygq-qwlvg sleqamldalvmdrvafv~_ ~smhkfltiprleelyntkqgptnpmlfh-lvrdvkqgn Glycosyl-H20asel active site Zn Bind-COase TRPM6 11s-gyri tlidiglvveyl igrayr~DYJ:khfralynnlyrkykhqrh-c5gnr~~est-lh-JlLI~kfk-ek TRPM7 l-ppgykitlidiglvieylmggtyrctytrkrfrliyn~l----ggnnrr-sg-~ lstpqlrk~he~ fgnr-adkkek PKcA TRPM6 ------si--vlhks'rkk keq-~ddpestgflypyndllvwavlmkrqkmamffwqhgeeatvkaviacilyrama TRPM7 mrhnhfiktaqpypkidtvme--egkkkr kdeivdiddpetkrfpyplnelliwaclml ...., TRPM6 rknr"wl kiii';i i1 TRPM7 rkn'wykvlls negkn-emeiqmk kklp TRPM6 wtr e 1"1'-.,....1 Y 1V'1" .... 'v ...... 'tl~i"'-""rzl'rll"1n.II'ynl it I" J ~ , J ":-:1""': ":. ::. "4;f J ~: •• '! ,"-A, ..... ,d TRPM7 r '"p: :.": .. , .. .. - TRPM 1 TRPM 1 TRPM6lf!=='1=PPSWSlard~ vfepy~e:dvc..,~q---~p-~5CP g'>fl tpflqavyl fvqy~ imvnlliaffn vyld TRPM7 ('1' ' :Hv apswtlakdlVfhpYWITllfgevyayeldvca-rutJlpql -cg twlt fl av ltv "mvnlllaffn vylq G-p sig TRPM version of TRP Domain TRPM6 me nnlwkynryry~mtyhekpwlpppllllshvglllrrl-cc--hraphdqeegdvglklylskedlkklhdfeeqcvekyfhekmed TRPM7 vkaisnlvwkyqryhfimayhekpvlppplil1shl-VS - Ifcclckrrkkdk-tsd-gpklflteedqkklhdfeeqcvemyfnekddk G-prot sig CPSase PKcA TRPM6 VlK ceeC-:L ~" ,-- . ~',1 .,; ','~ '~ haltvdtlkvbavd lqedeallakrkh Lckklph~w5nv TRPM7 fh g eer'r f' ",,' .~., key drvn I f>. ~,l~,': :1' lsaltvdtlktlLa------G-prot sig -ATP DNAase in Coiled-Coil Non-identical Serine, Threonine &Proline Rich.__., TRPM6 lcaevlg~melagekkyqyy mp llr' laggrhppr EU1~IGa~e~1P'~i;'~'~i~,i...i,,~~;r~i,~iIe TRPM7 ------ka:ea~Jc\'hnii . l~lt;tdtl: Phosphopantetheine Att Coiled-coil TRPM6 ~nrgah kyggf it' P nlkr•..Pf':>aetvlpl~rp~vpdvlateqd;qtevlvhl tgqtpvv5dwa~vdepkekhepl ahlldgqdkaeqgp TRPM7 kkhe:vvntls~ Ipqgdle~nnpfhcnilmkddkdpqcnifgqdlpavpg[kefnfgeag ~1.m~\5QQelrqrlhgvell Glyc-H20ase 11 TRPM6 vlptlJctpepmtmssplsqaklmqtgggyvnwafsegdetgvf1ikkkwqtclpstcdsd~5rseqhqkqaqdsslsdn cr~aq ~ecsev TRPM7 kifnknqklg~stsiphl ,spptkffv ~p5qp5ck~hletgtkdqetvc5kategdntefgafvghrd5mdlqrfketsnkik CJQ PKs ATP-bind region sig PkcA TRPM6 wlqplJlwi nplrryrpfarshsfrfhkeeklmki cki knlsg '>e•...gggaw,JgJsnH kdrrl kkkkn qglqvpi i tvnacsqsdqlnp TRPM7 ilsnnn entl------krv~slagftdchrtsipvhskqaekicIIQtedthevdskaalipdwlqd PKcA TRPM6 epgens i -seeeysknwftvskfshtgvepyi hqkmktkei gqcai qi sdylkqsqedlskns lwnsrs tnlDI.O!>11k" I g,ldk I as1 TRPM7 rpsnrempsee-gtln-gltspf------Kinase Region CysPase-Inh TRPM6 kspqephhhysai ernnlmrlsqti pftp',q ~ fageel \Jyrleessplnldksmsswsqrgraami qvlsreemdggl rkamrvvstwse TRPM7 koamd· nv\,,\ysavernnlmrlsqslpftpvpp- rgepv tvyrlee')spni InnsmsswsqlgrcaJt-fefr"Keemgggl rravkvqctwse Kinase Deletion begins Pyrro-C02 peptidase TRPM6 ddilkpgqvfivksflpevvrtwhkifqestvlhlclreiqqqraaqkliytfnqvkpqtipytprflevfliych~anqwltieky TRPM7 hdilk~ghlyiilsflpevvntwssiykedtvlhlclreiqqqraaqkltfafnqmkpksipysprflevfllych agqwfaveec Kringle TRPM6 mtgefrkynnnngdeitptntleelmlafshwtyeytrgellvldlqgvgenltdpsvikpevkqsrgmvf~panlgedalrnf TRPM7 mtgefrkynnnngdeiiptntleeimlafshwtyeytrgellvldlqgvgenltdpsvikaeekrscdmv~panlgedaiknf Heavy-Metal (Zn)Domain TRPM6 lakhhcn ccrk k~pdlkrndy perln 19:9jkiesaeepparet-grnspeddmql TRPM7 rakhhcnsccrklklpdlkrndytpdklifpqdepsdlnlqpin ~esestnsvrl-m-l ::r 1 APPENDIX III: DEFINITION OF MOTIFS FROM PROSITE DATABASE BY THE SWISS INSTITUTE OF BIOINFORMATICS NPS@ is the IBCP contribution to PBIL in Lyon, France NPS@: Network Protein Sequence Analysis Combet c., Blanchet C., Geomjon C. and Deleage G. TIBS 2000 March Vol. 25, No 3 [291]:147-150 PROSITE : Bairoch A., Bucher P. and Hofmann K. The PROSITE database, its status in 1997 Nucleic Acids Res. (1997) Jan 1; 25(1):217-221 PROSCAN : PROSCAN has been developed at IBCP. Protein trpm7xO & trpm6xO using PROSITE.BASE as reference site file Similarity percentage 80 ~------~ These PROSITE entries are copyright by the Swiss Institute of Bioinformatics (SIB). There are no restrictions on its use by non-profit institutions as long as its content is in no way modified and this statement is not removed. Usage by and for commercial entities requires a license agreement (See http://www.isb-sib.ch/announce/ or email to [email protected]). ~------~ Content was not modified except condensing blank spaces to save space. ************************ * N-glycosylation site * ************************ It has been known for a long time [1] that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. It must be noted that the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation [2]. It has been shown [3] that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent [4] statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. It must also be noted that there are a few reported cases of glycosylation sites with the pattern Asn-Xaa-Cys; an experimentally demonstrated occurrence ofsuch a non-standard site is found in the plasma protein C[5]. Last update:May 1991/Text Revised. -Consensus pattern: N-{P}-[ST]-{P} [N is the glycosylation site] [ 1] Marshall R.D. Annu. Rev. Biochem. 41:673-702(1972). [2] Pless D.D., Lennarz W.J. Proc. Natl. Acad. Sci. U.S.A. 74:134-138(1977). [3] Bause E. Biochem. J. 209:331-336(1983). [ 4] Gavel Y., von Heijne G. Protein Eng. 3:433-442(1990). [ 5] Miletich J.P., Broze G.J. Jr. J. BioI. Chern. 265:11397-11404(1990). j **************************************************************** * cAMP- and cGMP-dependent protein kinase phosphorylation site * **************************************************************** There has been a number of studies relative to the specificity of cAMP- and cGMP dependent protein kinases [1,2,3]. Both types ofkinases appear to share a preference for the phosphorylation of serine or threonine residues found close to at least two consecutive N-terminal basic residues. It is important to note that there are quite a number ofexceptions to this rule.- Last update: June 1988/ First entry -Consensus pattern: [RK](2)-x-[ST] [S or T is the phosphorylation site] [ I] Fremisco J.R., Glass D.B., Krebs E.G. J. BioI. Chern. 255:4240-4245(1980). [2] Glass D.B., Smith S.B. 1. BioI. Chern. 258:14797-14803(1983). [ 3] Glass D.B., EI-Maghrabi M.R., Pilkis S.J. J. BioI. Chern. 261 :2987-2993(1986). ***************************************** * Protein kinase C phosphorylation site * ***************************************** In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues found close to a C-terminal basic residue [1,2]. The presence of additional basic residues at the N- or C-terminal of the target amino acid enhances the Vmax and KIn ofthe phosphorylation reaction. -Last update: June 1988/ First entry -Consensus pattern: [ST]-x-[RK] [S or T is the phosphorylation site] [ 1] Woodget 1.R., Gould K.L., Hunter T. Eur. J. Biochem. 161:177-184(1986). [ 2] Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H.,Takeyama Y, Nishizuka Y 1. BioI. Chern. 260:12492-12499(1985). ***************************************** * Casein kinase II phosphorylation site * **************************************** Casein kinase II (CK-2) is a protein serine/threonine kinase whose activity is independent of cyclic nucleotides and calcium. CK-2 phosphorylates many different proteins. The substrate specificity [1] ofthis enzyme can be summarized as follows: (1) Under comparable conditions Ser is favored over Thr. (2) An acidic residue (either Asp or Glu) must be present three residues from the C-terminal ofthe phosphate acceptor site. (3) Additional acidic residues in positions +1, +2, +4, and +5 increase the phosphorylation rate. Most physiological substrates have at least one acidic residue in these positions. (4) Asp is preferred to Glu as the provider of acidic determinants. (5) A basic residue at the N-terminal of the acceptor site decreases the phosphorylation rate, while an acidic one will increase it. -Last update: May 1991 / Text revised. -Consensus pattern: [ST]-x(2)-[DE] [S or T is the phosphorylation site] -Note: this pattern is found in most ofthe known physiological substrates. [ 1] Pinna L.A. Biochim. Biophys. Acta 1054:267-284(1990). k **************************************** * Tyrosine kinase phosphorylation site * **************************************** Substrates of tyrosine protein kinases are generally characterized by a lysine or an arginine seven residues to the N-terminal side of the phosphorylated tyrosine. An acidic residue (Asp or Glu) is often found at either three or four residues to the N terminal side of the tyrosine [1,2,3]. There are a number ofexceptions to this rule such as the tyrosine phosphorylation sites ofenolase and lipocortin II. -Last update: June 1988 / First entry. -Consensus pattern: [RK]-x(2)-[DE]-x(3)-Y or [RK]-x(3)-[DE]-x(2)-Y [Y is the P site] [ 1] Patschinsky T., Hunter T., Esch F.S., Cooper J.A., Sefton B.M. PNAS 79:973 977(1982). [2] Hunter T. J. BioI. Chern. 257:4843-4848(1982). [ 3] Cooper lA., Esch F.S.,Taylor S.S.,Hunter T.J. BioI.Chem.259:7835-7841(1984). ************************* * N-myristoylation site * ************************* An appreciable number of eukaryotic proteins are acylated by the covalent addition of myristate (a Cl4-saturated fatty acid) to their N-terminal residue via an amide linkage [1,2]. The sequence specificity ofthe enzyme responsible for this modification, myristoyl CoA:protein N-myristoyl transferase (NMT), has been derived from the sequence of known N-myristoylated proteins and from studies using synthetic peptides. It seems to be the following: - The N-terminal residue must be glycine. - In position 2, uncharged residues are allowed. Charged residues, proline and large hydrophobic residues are not allowed. - In positions 3 and 4, most, ifnot all, residues are allowed. - In position 5, small uncharged residues are allowed (Ala, Ser, Thr, Cys, Asn and Gly). Serine is favored. - In position 6, proline is not allowed. -Consensus pattern: G-{EDRKHPFYW}-x(2)-[STAGCN]-{P} [G is the N-myr site] -Note: we deliberately include as potential myristoylated glycine residues, those which are internal to a sequence. It could well be that the sequence under study represents a viral polyprotein precursor and that subsequent proteolytic processing could expose an internal glycine as the N-terminal ofa mature protein. -Last update: October 1989 / Pattern and text revised. [ 1] TowlerD.A.,GordonJ.I.,AdamsS.P.,GlaserL. Annu. Rev.Biochem.57:69-99(1988). [2] Grand RJ.A. Biochem. J. 258:625-638(1989). ****************** * Amidation site * ****************** The precursor ofhormones and other active peptides which are C-terminally amidated is always directly followed [1,2] by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Valor Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. C-terminal amidation has not yet been shown to occur in unicellular organisms or in plants. -Last update: June 1988 / First entry. -Consensus pattern: x-G-[RK]-[RK] [x is the amidation site] [ 1] Kreil G. Meth. Enzymol. 106:218-223(1984). [2] Bradbury A.F., Smyth D.G. Biosci. Rep. 7:907-916(1987). ************************************** * Phosphopantetheine attachment site * ************************************** Phosphopantetheine (or pantetheine 4' phosphate) is the prosthetic group of acyl carrier proteins (ACP) in some multienzyme complexes where it serves as a 'swinging arm' for the attachment of activated fatty acid and amino-acid groups[l]. Phosphopantetheine is attached to a serine residue in these proteins [2]. ACP proteins or domains have been found in various enzyme systems which are listed below (references are only provided for recently determined sequences). - Fatty acid synthetase (FAS), which catalyzes the formation oflong-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH. Bacterial and plant chloroplast FAS are composed of eight separate subunits which correspond to the different enzymatic activities; ACP is one of these polypeptides. Fungal FAS consists of two multifunctional proteins, FASI and FAS2; the ACP domain is located in the N terminal section of FAS2. Vertebrate FAS consists of a single multifunctional enzyme; the ACP domain is located between the beta-ketoacyl reductase domain and the C-terminal thioesterase domain [3]. - Polyketide antibiotics synthase enzyme systems. Polyketides are secondary metabolites produced from simple fatty acids, by microorganisms and plants. ACP is one of the polypeptidic components involved in the biosynthesis of Streptomyces polyketide antibiotics actinorhodin, curamycin, granatacin, monensin, oxytetracycline and tetracenomycin C. - Bacillus subtilis putative polyketide synthases pksK, pksL and pksM, which respectively contain three, five and one ACP domains. - The multifunctional 6-methysalicylic acid synthase (MSAS) from Penicillium patulum. This is a multifunctional enzyme involved in the biosynthesis of a polyketide antibiotic and which contains an ACP domain in the C-terminal extremity. - Multifunctional mycocerosic acid synthase (gene mas) from Mycobacterium bovis. - Gramicidin S synthetase I (gene grsA) from Bacillus brevis. This enzyme catalyzes the first step in the biosynthesis of the cyclic antibiotic gramicidin S. m - Tyrocidine synthetase I (gene tycA) from Bacillus brevis. The reaction carried out by tycA is identical to that catalyzed by grsA - Gramicidin S synthetase II (gene grsB) from Bacillus brevis. This enzyme is a multifunctional protein that activates and polymerizes proline, valine, ornithine and leucine. GrsB contains four ACP domains. - Erythronolide synthase proteins 1, 2 and 3 from Saccharopolyspora erythraea which is involved in the biosynthesis of the polyketide antibiotic erythromicin. Each ofthese proteins contain two ACP domains. - Conidial green pigment synthase from Aspergillus nidulans. - ACV synthetase from various fungi. This enzyme catalyzes the first step in the biosynthesis of penicillin and cephalosporin. It contains three ACP domains. - Enterobactin synthetase component F (gene entF) from Escherichia coli. This enzyme is involved in the ATP-dependent activation of serine during enterobactin (enterochelin) biosynthesis. - Cyclic peptide antibiotic surfactin synthase subunits 1, 2 and 3 from Bacillus subtilis. Subunits 1 and 2 contains three related domains while subunit 3 only contains a single domain. - HC- toxin synthetase (gene HTS1) from Cochliobolus carbonum. This enzyme synthesizes HC-toxin, a cyclic tetrapeptide. HTS1 contains four ACP domains. - Fungal mitochondrial ACP, which is part ofthe respiratory chain NADH dehydrogenase (complex I). - Rhizobium nodulation protein nodF, which probably acts as an ACP in the synthesis ofthe nodulation Nod factor fatty acyl chain. The sequence around the phosphopantetheine attachment site is conserved in all these proteins and can be used as a signature pattern. A profile was also developed that spans the complete ACP-like domain. -Consensus pattern: [DEQGSTALMKRH]-[LIVMFYSTAC]-[GNQ]-[LIVMFYAG] [DNEKHS]-S-[LIVMST]-{PCFY}-[STAGCPQLIVMF]-[LIVMATN] {DENQGTAKRHLM]-[LIVMWSTA]-[LIVGSTACR]-x(2)-[LIVMFA] [8 is the pantetheine attachment site] -Sequences known to belong to this class detected by the pattern: ALL, except paradoxa ACP. Other sequence(s) detected in Swiss-Prot: 115. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: November 1997/ Pattern and text revised; profile added. [1] Concise Encyclopedia Biochemistry, 2nd Edition, Walter de Gruyter, Berlin New York (1988). [2] Pugh E.L., Wakil SJ. J. BioI. Chern. 240:4727-4733(1965). [3] Witkowski A., Rangan V.S., Randhawa Z.I., Amy C.M., Smith S. Eur. J. Biochem. 198:571-579(1991). n ********************************** * EF-hand calcium-binding domain * ********************************** Many calcium-binding proteins belong to the same evolutionary family and share a type ofcalcium-binding domain known as the EF-hand [1 to 5]. This type ofdomain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand). We list below the proteins which are known to contain EF-hand regions. For each type of protein we have indicated between parenthesis the total number of EF-hand regions known or supposed to exist. This number does not include regions which clearly have lost their calcium-binding properties, or the atypical low-affinity site (which spans thirteen residues) found in the S-lOO/ICaBP family ofproteins [6]. - Aequorin and Renilla luciferin binding protein (LBP) (Ca=3). Alpha actinin (Ca=2). Calbindin (Ca=4). Calcineurin B subunit (protein phosphatase 2B regulatory subunit) (Ca=4). Calcium-binding protein from Streptomyces erythraeus (Ca=3?). Calcium binding protein from Schistosoma mansoni (Ca=2?). Calcium-binding proteins TCBP 23 and TCBP-25 from Tetrahymena thermophila (Ca=4?). Calcium-dependent protein kinases (CDPK) from plants (Ca=4). Calcium vector protein from amphoxius (Ca=2). Calcyphosin (thyroid protein p24) (Ca=4?). Calmodulin (Ca=4, except in yeast where Ca=3). Calpain small and large chains (Ca=2). Calretinin (Ca=6). Calcyclin (prolactin receptor associated protein) (Ca=2). Caltractin (centrin) (Ca=2 or 4). Cell Division Control protein 31 (gene CDC31) from yeast (Ca=2?). Diacylglycerol kinase (EC 2.7.1.107) (DGK) (Ca=2). FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) from mammals (Ca=I). Fimbrin (plastin) (Ca=2). Flagellar calcium-binding protein (1f8) from Trypanosoma cruzi (Ca=1 or 2). Guanylate cyclase activating protein (GCAP) (Ca=3). Inositol phospholipid-specific phospholipase C isozymes gamma-l and delta-l (Ca=2) [10]. Intestinal calcium-binding protein (ICaBPs) (Ca=2). MIF related proteins 8 (MRP-8 or CFAG) and 14 (MRP-14) (Ca=2). Myosin regulatory light chains (Ca=I). Oncomodulin (Ca=2). Osteonectin (basement membrane protein BM-40) (SPARC) and proteins that contains an 'osteonectin' domain (QR1, matrix glycoprotein SCI) (see the entry A common feature of all EGF-like domains is that they are found in the extracellular domain of membrane-bound proteins or in proteins known to be secreted (exception: prostaglandin G/H synthase). The EGF-like domain includes six cysteine residues which have been shown to be involved in disulfide bonds. The structure of several EGF-like domains has been solved. The fold consists of two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet (see The two patterns we developed recognize both superfamilies. Our first pattern recognizes the proximal heme-binding site whereas the second pattern surrounded the distal r active site. We also developed two profiles, one specific for the animal peroxidases superfamily and one directed against the plant peroxidase superfamily. -Consensus pattern:[DET]-[LIVMTA]-x(2)-[LIVM]-[LIVMSTAG]-[SAG] [LIVMSTAG]-H-[STA]-[LIVMFY] [H is the proximal heme-binding ligand] Sequences known to belong to this class detected by the profile: ALL. for ligninase III from Phlebia radiata, and LPO. Other sequence(s) detected in Swiss-Prot: NONE. -Consensus pattern: [SGATV]-x(3)-[LIVMA]-R-[LIVMA]-x-[FW]-H-x-[SAC] [H is an active site residue] -Sequences known to belong to this class detected by the profile: ALL. for vertebrate peroxidases (MPO, TPO, LPO, and EPa). Other sequence(s) detected in Swiss-Prot: NONE. Sequences known to belong to this class detected by the first profile: ALL. Other sequence(s) detected in Swiss~Prot: NONE. Sequences known to belong to this class detected by the second profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: November 2002/ Text revised; profiles added. [ 1] Dawson J.H. Science 240:433-439(1988). [ 2] Kimura S., Ikeda-Saito M. Proteins 3: 113-120(1988). [ 3] Henrissat B., Saloheimo M., Lavaitte S., Knowles J.K.C. Proteins 8:251- 257(1990). [ 4] Welinder K.G. Biochim. Biophys. Acta 1080:215-220(1991). **************************************** * Kringle domain signature and profile * **************************************** Kringles [1,2,3] are triple-looped, disulfide cross-linked domains found in a varying number of copies, in some serine proteases and plasma proteins. The kringle domain has been found in the following proteins: Apolipoprotein A (38 copies). Blood coagulation factor XII (Hageman factor) (l copy). Hepatocyte growth factor (HGF) (4 copies). Hepatocyte growth factor like protein (4 copies) [4]. Hepatocyte growth factor activator [1] (once) [5]. Plasminogen (5 copies). Thrombin (2 copies). Tissue plasminogen activator (TPA) (2 copies). Urokinase-type plasminogen activator (l copy). The schematic representation ofthe structure ofa typical kringle domain is shown below: ~------~ I I xCxxxxxxxxxxxCxxxxxxxxxxCxxxxxCxxxxxxCxxxCx I ~------I------~ I ~------~ 'C': conserved cysteine involved in a disulfide bond. Kringle domains are thought to play a role in binding mediators, such as membranes, other proteins or phospholipids, and in the regulation of proteolytic activity. As a signature pattern for this type of domain, we selected a conserved sequence that contains two ofthe cysteines invovled in disulfide bonds. -Consensus pattern: [FY]-C-[RH]-[NS]-x(7,8)-[WY]-C s [The 2 CIS are involved in a disulfide bonds] - Expert(s) to contact by email: IkeoK.;[email protected] -Last update: May 2004 I Text revised. [ 1] Castellino FJ., Beals J.M. J. Mol. Evol. 26:358-369(1987). [2] Patthy L. Cell 41:657-663(1985). [3] Ikeo K., Takahashi K., Gojobori T. FEBS Lett. 287:146-148(1991). [ 4] Friezner Degen S.J., Stuart L.A., Han S., Jamison C.S.Biochemistry 30:9781 9791(1991). [ 5] Miyazawa K., Shimomura T., Kitamura A, Kondo J., Morimoto Y., Kitamura N. J. BioI. Chern. 268:10024-10028(1993). ************************************************* * N-6 Adenine-specific DNA methylases signature * ************************************************* N-6 adenine-specific DNA methy1ases (EC 2.1.1.72) (A-Mtase) are enzymes that specifically methylate the amino group at the C-6 position of adenines in DNASuch enzymes are found in the three existing types of bacterial restriction-modification systems (in type I system the A-Mtase is the product ofthe hsdM gene, and in type III it is the product of the mod gene). All of these enzymes recognize a specific sequence in DNA and methylate an adenine in that sequence. It has been shown [1,2,3,4] that A Mtases contain a conserved motifAsplAsn-Pro-Pro-Tyr/Phe in their N-terminal section, this conserved region could be involved in substrate binding or in the catalytic activity. We have derived a pattern from that motif. -Consensus pattern: [LIVMAC]-[LIVFYWA]-x-[DN]-P-P-[FYW] -Sequences known to belong to this class detected by the pattern: ALL, except for m.HhaII where the second Pro is replaced by GIn and in m.HindIII where that same Pro is replaced by Tyr. Other sequence(s) detected in Swiss-Prot: 33 different proteins that are most probably not A-Mtases, and three hypothetical Escherichia coli proteins that could be A-Mtases. -Note: N-4 cytosine-specific DNA methylases, which are probably enzymatically related to A-Mtases, also include a conserved Pro-Pro dipeptide but the residues around them are sufficiently different to allow the derivation ofa pattern specific to these enzymes. -Expert(s) to contact by email: Roberts R.J.; [email protected] Bickle T.; [email protected] -Last update: November 1997 I Text revised. [ 1] Loenen W.AM., Daniel A.S., Braymer H.D., Murray N.E. J. Mol. BioI. 198:159 170(1987). [2] Narva K.E., van Etten J.L., Slatko B.E., Benner 1.S. Gene 74:253-259(1988). [ 3] Lauster R. 1. Mol. BioI. 206:313-321(1989). [4] Timinskas A, Butkus V., Janulaitis A Gene 157:3-11(1995). ********************************************** * C-type lectin domain signature and profile * ********************************************** A number of different families of proteins share a conserved domain which was first characterized in some animal lectins and which seem to function as a calcium- t dependent carbohydrate-recognition domain [1,2,3]. This domain, which is known as the C-type lectin domain (CTL) or as the carbohydrate-recognition domain (CRD), consists of about 110 to 130 residues. There are four cysteines which are perfectly conserved and involved in two disulfide bonds. A schematic representation ofthe CTL domain is shown below. +------+ II xcxxxxcxxxxxxxCxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxCxxxxVVxCxxxxCx I ************1* +----+ +------+ 'C': conserved cysteine involved in a disulfide bond. 'c': optional cysteine involved in a disulfide bond. '*': position ofthe pattern. The categories ofproteins, in which the CTL domain has been found, are listed below. Type-II membrane proteins where the CTL domain is located at the C-terminal extremity of the proteins: Asialoglycoprotein receptors (ASGPR) (also known as hepatic lectins) [4]. The ASGPR's mediate the endocytosis of plasma glycoproteins to which the terminal sialic acid residue in their carbohydrate moieties has been removed. Low affinity immunoglobulin epsilon Fc receptor (lymphocyte IgE receptor),which plays an essential role in the regulation of IgE production and in the differentiation of B cells. Knpffer cell receptor. A receptor with an affinity for galactose and fucose, that could be involved in endocytosis. A number of proteins expressed on the surface of natural killer T-cells: NKG2, NKR-Pl, YE1I88 (Ly-49), CD69 and on B-cells: CD72, LyB-2. The CTL-domain in these proteins is distantly related to other CTL-domains; it is unclear whether they are likely to bind carbohydrates. Proteins that consist ofan N-terminal collagenous domain followed by a CTL- domain [5], these proteins are sometimes called 'collectins': Pulmonary surfactant associated protein A (SP-A). SP-A is a calcium-dependent protein that binds to surfactant phospholipids and contributes to lower the surface tension at the air-liquid interface in the alveoli of the mammalian lung. Pulmonary surfactant-associated protein D (SP-D). Conglutinin, a calcium-dependent lectin-like protein which binds to a yeast cell wall extract and to immune complexes through the complement component (iC3b). Mannan-binding proteins (MBP) (also known as mannose-binding proteins). MBP's bind mannose and N-acetyl-D-glucosamine in a calcium-dependent manner. Bovine collectin-43 (CL-43). Selectins (or LEC-CAM) [6,7]. Selectins are cell adhesion molecules implicated in the interaction of leukocytes with platelets or vascular endothelium. Structurally, selectins consist ofa long extracellular domain, followed by a transmembrane region and a short cytoplasmic domain. The extracellular domain is itself composed of a CTL-domain, followed by an EGF-like domain and a variable number of SCR/Sushi repeats. Known selectins are: Lymph node homing receptor (also known as L-selectin, leukocyte adhesion molecule-I, (LAM-I), leu-8, gp90-mel, or LECAM-l). Endothelial leukocyte adhesion molecule 1 (ELAM-l, E-selectin or LECAM-2). The ligand recognized by ELAM-l is sialyl-Lewis x. Granule membrane protein 140 (GMP-140, P-selectin, PADGEM, CD62, or LECAM-3). The ligand recognized by GMP-140 is Lewis x. u Large proteoglycans that contain a CTL-domain followed by one copy ofa SCRf Sushi repeat, in their C-terminal section: Aggrecan (cartilage-specific proteoglycan core protein). This proteoglycan is a major component of the extracellular matrix of cartilagenous tissues where it has a role in the resistance to compression. Brevican. Neurocan. Versican (large fibroblast proteoglycan), a large chondroitin sulfate proteoglycan that may playa role in intercellular signalling. In addition to the CTL and Sushi domains, these proteins also contain, in their N-terminal domain, an Ig-like V type region, two or four link domains (see As a signature pattern for this domain, we selected the C-terminal region with its three conserved cysteines. -Consensus pattern: C-[LIVMFYATG]-x(5,12)-[WL]-x-[DNSR]-x(2)-C-x(5,6)- [FYWLIVSTA]-[LIVMSTA]-C [The 3 C's are involved in disulfide bonds] v -Sequences known to belong to this class detected by the pattern: ALL, except the distantly related natural killer T-cell and B-cell proteins. Other sequence(s) detected in Swiss-Prot: 15. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: 2. -Note: all CTL domains have five Trp residues before the second Cys, with the exception oftunicate lectin and cockroach LPS-BP which have Leu. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. Expert(s) to contact by email: DrickamerK.;[email protected] -Last update: May 2004 / Text revised. [ 1] Drickamer K. J. BioI. Chern. 263:9557-9560(1988). [2] Drickamer K. Prog. Nucleic Acid Res. Mol. BioI. 45:207-232(1993). [3] Drickamer K. Curr. Opin. Struct. BioI. 3:393-400(1993). [4] Spiess M. Biochemistry 29:10009-10018(1990). [ 5] Weis W.I., Kahn R., Fourme R., Drickamer K., Hendrickson W.A. Science 254: 1608-1615(1991). [6] Siegelman M. Curr. BioI. 1:125-128(1991). [ 7] Lasky L.A. Science 238:964-969(1992). [8] Jomori T., Natori S. 1. BioI. Chern. 266:13318-13323(1991). [9] Ng N.F.L., Hew c.-L. 1. BioI. Chern. 267:16069-16075(1992). ******************************************* * 'Homeobox' domain signature and profile * ******************************************* The 'homeobox' is a protein domain of 60 amino acids [1 to 5,E1] first identified in a number ofDrosophila homeotic and segmentation proteins. It has since been found to be extremely well conserved in many other animals, including vertebrates. This domain binds DNA through a helix-turn-helix type of structure. Some of the proteins which contain a homeobox domain play an important role in development. Most of these proteins are known to be sequence specific DNA-binding transcription factors. The homeobox domain has also been found to be very similar to a region of the yeast mating type proteins. These are sequence-specific DNA-binding proteins that act as master switches in yeast differentiation by controlling gene expression in a cell type specific fashion. A schematic representation of the homeobox domain is shown below. The helix-turn helix region is shown by the symbols 'H' (for helix), and It' (for turn). xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxHHHHHHHHtttHHHHHHHHHxxxxxxxxxx I I I 1 10 20 30 40 50 60 The pattern we developed to detect homeobox sequences is 24 residues long and spans positions 34 to 57 ofthe homeobox domain. -Consensus pattern: [LIVMFYG]-[ASLVR]-x(2)-[LIVMSTACN]-x-[LIVM]-x(4)-[LIV] [RKNQESTAIY]-[LIVFSTNKH]-W-[FYVC]-x-[NDQTAH]-x(5)-[RKNAIMW] w -Sequences known to belong to this class detected by the pattern: ALL, except for 10 sequences. Other sequence(s) detected in Swiss-Prot: 9. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: proteins which contain a homeobox domain can be classified, on the basis of their sequence characteristics, into various subfamilies. We have developed specific patterns for conserved elements ofthe antennapedia, engrailed and paired families. -Note: this documentation entry is linked to both signature patterns and a profile. As the profile is much more sensitive than the patterns, you should use it ifyou have access to the necessary software tools to do so. -Expert(s) to contact by email: BuerglinT.R.;[email protected] -Last update: July 1998/ Pattern and text revised. [ 1] Gehring W.J. (In) Guidebook to the homebox genes, Duboule D., Ed., ppl-IO, Oxford University Press, Oxford, (1994). [ 2] Buerglin T.R. (In) Guidebook to the homebox genes, Duboule D., Ed., pp25-72, Oxford University Press, Oxford, (1994). [3] Gehring W.J. Trends Biochem. Sci. 17:277-280(1992). [4] Gehring W.J., Hiromi Y. Annu. Rev. Genet. 20:147-173(1986). [ 5] Schofield P.N. Trends Neurosci. 10:3-6(1987). [E1] http://www.biosci.ki.se/groups/tbu/homeo.html *********************************** * Ribosomal protein S2 signatures * *********************************** Ribosomal protein S2 is one of the proteins from the small ribosomal subunit. S2 belongs to a family of ribosomal proteins which, on the basis of sequence similarities [1,2], groups: - Eubacterial S2. - Algal and plant chloroplast S2. - Cyanelle S2. - Archaebacterial S2. Higher eukaryotes P40 (previously thought to be a laminin receptor). - Yeast NAB1. Plant mitochondrial S2. - Yeast mitochondrial MRP4. S2 is a protein of 235 to 394 amino-acid residues. As signature patterns, we selected two conserved regions. One is located in the N-terminal section and the other in the central section. -Last update: November 1997 / Patterns and text revised. -Consensus pattern: [LIVMFA]-x(2)-[LIVMFYC](2)-x-[STAC]-[GSTANQEKR] [STALV]-[HY]-[LIVMF]-G -Sequences known to belong to this class detected by the pattern: ALL, exceptfor three sequences. Other sequence(s) detected in Swiss-Prot: 41. -Consensus pattern: P-x(2)-[LIVMF](2)-[LIVMS]-x-[GDN]-x(3)-[DENL]-x(3)-[LIVM]- x-E-x(4)-[GNQKRH]-[LIVM]-[AP] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Last update: November 1997 / Patterns and text revised. [1] Davis S.C., TzagoloffA., Ellis S.R. 1. BioI. Chern. 267:5508-5514(1992). [2] Tohgo A., Takasawa S., Munakata H., Yonekura H., Hayashi N., Okamoto H. FEBS Lett. 340:133-138(1994). x ****************************************** * Protein kinases signatures and profile * ****************************************** Eukaryotic protein kinases [1 to 5] are enzymes that belong to a very extensive family of proteins which share a conserved catalytic core common to both serine/threonine and tyrosine protein kinases. There are a number of conserved regions in the catalytic domain of protein kinases. We have selected two of these regions to build signature patterns. The first region, which is located in the N-terrninal extremity of the catalytic domain, is a glycine-rich stretch ofresidues in the vicinity of a lysine residue, which has been shown to be involved in ATP binding. The second region, which is located in the central part of the catalytic domain, contains a conserved aspartic acid residue which is important for the catalytic activity of the enzyme [6]; we have derived two signature patterns for that region: one specific for serine/ threonine kinases and the other for tyrosine kinases. We also developed a profile which is based on the alignment in [1] and covers the entire catalytic domain. -Consensus pattern:[LIV]-G- {P} -G- {P}-[FYWMGSTNH]-[SGA]-{PW} -[LIVCAT] {PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]-[LIVMFAGCKR]-K [K binds ATP] -Sequences known to belong to this class detected by the pattern: the majority ofknown protein kinases but it fails to find a number ofthem, especially viral kinases which are quite divergent in this region and are completely missed by this pattern. -Consensus pattern: [LIVMFYC]-x-[HY]-x-D-[LIVMFY]-K-x(2)-N-[LIVMFYCT](3) [D is an active site residue] -Sequences known to belong to this class detected by the pattern: Most serine/ threonine specific protein kinases with 10 exceptions (half of them viral kinases) and also Epstein-Barr virus BGLF4 and Drosophila ninaC which have respectively Ser and Arg instead of the conserved Lys and which are therefore detected by the tyrosine kinase specific pattern described below. - Cons ensus patte rn:[LIVMFYC]-x-[HY]-x-D-[LIVMFY]-[RSTAC]-x(2)-N- [LIVMFYC](3) [D is an active site residue] -Sequences known to belong to this class detected by the pattern: ALL tyrosine specific protein kinases with the exception of human ERBB3 and mouse blk. This pattern will also detect most bacterial aminoglycoside phosphotransferases [8,9] and herpesviruses ganciclovir kinases [10]; which are proteins structurally and evolutionary related to protein kinases. -Sequences known to belong to this class detected by the profile: ALL, except for three viral kinases. This profile also detects receptor guanylate cyclases (see One of the conserved regions in these enzymes is centered on a conserved glutamic acid residue which has been shown [5], in the beta-glucosidase from Agrobacterium, to be directly involved in glycosidic bond cleavage by acting as a nucleophile. We have used this region as a signature pattern. As a second signature pattern we selected a conserved region, found in the N-terminal extremity of these enzymes, this region also contains a glutamic acid residue. z -Consensus pattern: [LIVMFSTC]-[LIVFYS]-[LIV]-[LIVMST]-E-N-G-[LIVMFAR]- [CSAGN] [E is the active site residue] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 14 -Note: this pattern will pick up the last two domains of LPH; the first two domains, which are removed from the LPH precursor by proteolytic processing, have lost the active site glutamate and may therefore be inactive [4]. -Consensus pattern: F-x-[FYWM]-[GSTA]-x-[GSTA]-x-[GSTA](2)-[FYNH]-[NQ]-x-E x-[GSTA] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: this pattern will pick up the last three domains ofLPH. -Expert(s) to contact by email: HenrissatR;[email protected] -Last update: November 1995 / Patterns and text revised. [ 1] Henrissat R Biochem.1. 280:309-316(1991). [2] Henrissat R Protein Seq. Data Anal. 4:61-62(1991). [3] Gonzalez-Candelas L., Ramon D., Polaina J. Gene 95:31-38(1990). [ 4] EI Hassouni M., Henrissat B., Chippaux M., Barras F. J. Bacteriol. 174:765 777(1992). [ 5] Withers S.G., Warren R.A.J., Street J.P., Rupitz K., Kempton J.R, Aebersold R. J. Am. Chern. Soc. 112:5887-5889(1990). ********************************************************* * Glycosyl hydrolases family 11 active sites signatures * ********************************************************* The microbial degradation of cellulose and xylans requires several types of enzymes such as endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) (exoglucanases), or xylanases (EC 3.2.1.8) [1,2]. Fungi and bacteria produces a spectrum of cellulolytic enzymes (cellulases) and xylanases which, on the basis ofsequence similarities, can be classified into families. One of these families is known as the cellulase family G [3] or as the glycosyl hydrolases family 11 [4,El]. The enzymes which are currently known to belong to this family are listed below. -Aspergillus awamori xylanase C( xynC). Bacillus circulans, pumilus, stearothermophilus and subtilis xylanase (xynA). Clostridium acetobutylicum xylanase (xynB). Clostridium stercorarium xylanase A (xynA). Fibrobacter succinogenes xylanase C (xynC) which consist of two catalytic domains that both belong to family 10. Neocallimastix patriciarum xylanase A (xynA). Ruminococcus flavefaciens bifunctional xylanase XYLA (xynA). This protein consists of three domains: aN-terminal xylanase catalytic domain that belongs to family 11 of glycosyl hydrolases; a central domain composed of short repeats of GIn, Asn an Trp, and a C-terminal xylanase catalytic domain that belongs to family 10 of glycosyl hydrolases. Schizophyllum commune xylanase A. Streptomyces lividans xylanases B (xlnB) and C (xlnC). Trichoderma reesei xylanases I and II. -Two of the conserved regions in these enzymes are centered on glutamic acid residues which have both been shown [5], in Bacillus pumilis xylanase, to be necessary for catalytic activity. We have used both regions as signature patterns. -Last update: November 1997/ Text revised. aa -Consensus pattern: [PSA]-[LQ]-x-E-Y-Y-[LIVM](2)-[DE]-x-[FYWHN] [E is an active site residue] -Sequences known to belong to this class detected by the pattern: ALL, except for Piromyces sp. xynA. Other sequence(s) detected in Swiss-Prot: NONE. -Consensus pattern: [LIVMF]-x(2)-E-[AG]-[YWG]-[QRFGS]-[SG]-[STAN]-G-x-[SAF] [E is an active site residue] -Sequences known to belong to this class detected by the pattern: ALL, except for Piromyces sp. xynA. Other sequence(s) detected in Swiss-Prot: 3. -Expert(s) to contact by email: HenrissatB.;[email protected] -Last update: November 1997 / Text revised. [ 1] Beguin P. Annu. Rev. Microbiol. 44:219-248(1990). [ 2] Gilkes N.R., Henrissat B., Kilburn D.G., Miller R.C. Jr., Warren R.A.J. Microbiol. Rev. 55:303-315(1991). [ 3] Henrissat B., Claeyssens M., Tomme P., Lemesle L., Momon J.-P. Gene 81:83 95(1989). [4] Henrissat B. Biochem. J. 280:309-316(1991). [ 5] Ko E.P., Akatsuka H., Moriyama H., Shinmyo A., Hata Y., Katsube Y.,Urabe I., Okada H. Biochem. J. 288:117-121(1992). [E 1] http://www.expasy.org/cgi-bin/lists?glycosid.txt ************************************************** * Pyrrolidone-carboxylate peptidase active sites * ************************************************** Pyrrolidone-carboxylate peptidase (EC 3.4.19.3) (PYRase) (also known as pyroglutamyl peptidase) is the enzyme that selectively removes pyroglutamate (pGlu) from the N-terminus of proteins and peptides. In bacteria and archebacteria PYRase (gene pcp) is a protein of22-25 kD.1t is a cysteine protease with a Cys-His-Glu catalytic triad [1,2]. We developed two signature patterns that respectively include the glutamate and cysteine active site residues. -Consensus pattern: G-x(2)-[GAP]-x(4)-[LIV]-[ST]-x-E-[KR]-[LIVC]-[AG]-x-[NG] [E is the active site residue] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Consensus pattern: [LIVF]-x-[GSAV]-x-[LIVM]-S-x-[STAD]-A-G-x-[FY]-[LIV]-C- [DN] [C is the active site residue] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: these proteins belong to family C15 in the classification ofpeptidases [3,El]. -Last update: December 2001/ Pattern and text revised. [ 1] Singleton M.R., Isupov M.N., Littlechild 1.A. Structure 7:237-244(1999). [ 2] Odagaki Y., Hayashi A., Okada K., Hirotsu K., Kabashima T., Ito K. Yoshimoto T., Tsuru D., Sato M., Clardy 1. Structure 7:399-411(1999). [3] Rawlings N.D., Barrett A.1. Meth. Enzymol. 244:461-486(1994). [E 1] http://wvN/.expasy.org/cgi-bin/lists?peptidas.txt bb ******************************************** * Pancreatic ribonuclease family signature * ******************************************** Pancreatic ribonucleases (EC 3.1.27.5) are pyrimidine-specific endonucleases present in high quantity in the pancreas ofa number ofmammalian taxa and ofa few reptiles [1,2]. As shown in the following schematic representation ofthe sequence ofpancreatic RNases there are four conserved disulfide bonds and three amino acid residues involved in the catalytic activity. ~------~ ~------I------~ IIII xxxxx#xxxxxxCxxxxxxC#xxxxxxxCxxCxxxCxxxxxCxxxxxCxxxxxxCxxx#xxx **** I I ~------~ I ~------~ 'C': conserved cysteine involved in a disulfide bond. '#': active site residue. '*': position of the pattern. A number ofother proteins belongs to the pancreatic RNAse family and these are listed below. Bovine seminal vesicle and bovine brain ribonucleases. The kidney non secretory ribonucleases (also known as eosinophil-derived neurotoxin (EDN) [3]). Liver-type ribonucleases [4]. Angiogenin, which induces vascularization of normal and malignant tissues. It abolishes protein synthesis by specifically hydrolyzing cellular tRNAs. Eosinophil cationic protein (ECP) [5], a cytotoxin and helminthotoxin with ribonuclease activity. Frog liver ribonuclease and frog sialic acid-binding lectin [6]. The signature pattern we developed for these proteins includes five conserved residues: a cysteine involved in a disulfide bond, a lysine involved in the catalytic activity and three other residues important for substrate binding. -Consensus pattern: C-K-x(2)-N-T-F [C is involved in a disulfide bond] [K is an active site residue] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 4. Last update: October 1993/ Text revised. [ 1] Beintema 1.J., Schuller C., Irie M., Carsana A.Prog. Biophys. Mol. BioI. 51:165 192(1988). [2] Beintema J.1., van der Lann J.M. FEBS Lett. 194:338-343(1986). [3] Rosenberg H.F., Tenen D.G., Ackerman S.1. PNAS 86:4460-4464(1989). [ 4] Hofsteenge J., Matthies R., Stone S.R. Biochemistry 28:9806-9813(1989). [5] Rosenberg H.F., Ackerman S.l, Tenen D.G. J. Exp. Med. 170:163-176(1989). [ 6] Lewis M.T., Hunt L.T., Barker W.e. Protein Seq. Data Anal. 2:101-105(1989). ****************************************** * Serine carboxypeptidases, active sites * ****************************************** All known carboxypeptidases are either metallo carboxypeptidases or serine carboxypeptidases (EC 3.4.16.5 and EC 3.4.16.6). The catalytic activity of the serine carboxypeptidases, like that of the trypsin family serine proteases, is provided by a charge relay system involving an aspartic acid residue hydrogen-bonded to a histidine, cc which is itself hydrogen-bonded to a serine [l]. Proteins known to be serine carboxypeptidases are: Barley and wheat serine carboxypeptidases I, II, and III [2]. Yeast carboxypeptidase Y (YSCY) (gene PRC1), a vacuolar protease involved in degrading small peptides. Yeast KEX1 protease, involved in killer toxin and alpha-factor precursor processing. Fission yeast sxa2, a probable carboxypeptidase involved in degrading or processing mating pheromones [3]. Penicillium janthinellum carboxypeptidase S1 [4]. Aspergullus niger carboxypeptidase pepF. Aspergullus satoi carboxypeptidase cpdS. Vertebrate protective protein / cathepsin A [5], a lysosomal protein which is not only a carboxypeptidase but also essential for the activity of both beta-galactosidase and neuraminidase.Mosquito vitellogenic carboxypeptidase (VCP) [6]. Naegleria fowleri virulence-related protein Nf314 [7]. Yeast hypothetical protein YBR139w. Caenorhabditis elegans hypothetical proteins C08H9.1, F13DI2.6, F32A5.3, F41C3.5 and KIOB2.2. This family also includes: Sorghum (s)-hydroxymandelonitrile lyase (EC 4.1.2.11) (hydroxynitrile lyase) (HNL) [8], an enzyme involved in plant cyanogenesis. The sequences surrounding the active site serine and histidine residues are highly conserved in all these serine carboxypeptidases. -Consensus pattern: [LIVM]-x-[GSTA]-E-S-Y-[AG]-[GS] [S is the active site residue] -Sequences known to belong to this class detected by the pattern: ALL, except for HNL.Other sequence(s) detected in Swiss-Prot: 3. -Consensus pattern:[LIVF]-x(2)-[LIVSTA]-x-[IVPST]-x-[GSDNQL]-[SAGV]-[SG]-H- x- [IVAQ]-P-x(3)-[PSA] [H is the active site residue] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: these proteins belong to family SIO in the classification ofpeptidases [9,El]. -Last update: February 2003/ Patterns and text revised. [1] Liao D.l., Remington S.1. J. BioI. Chern. 265:6528-6531(1990). [2] Sorensen S.B., Svendsen 1., Breddam K. Carlsberg Res. Commun. 54:193-202(1989). [3] Imai Y, Yamamoto M. Mol. Cell. BioI. 12:1827-1834(1992). [ 4] Svendsen 1., Hofmann T., Endrizzi J., Remington J., Breddam K.FEBS Lett. 333:39 43(1993). [ 5] Galjart N.J., Morreau H., Willemsen R., Gillemans N., Bonten E.1., d'Azzo A. J. BioI. Chern. 266:14754-14762(1991). [6] Cho W.L., Deitsch K.W., Raikhel A.S. PNAS 88:10821-10824(1991). [7] Hu W.N., Kopachik W., Band R.N. Infect. Immun. 60:2418-2424(1992). [ 8] Wajant H., Mundry K.W., Pfitzenmaier K. Plant Mol. BioI. 26:735-746(1994). [9] Rawlings N.D., Barrett A.J. Meth. Enzymol. 244:19-61(1994). [E 1] http://www.expasy.org/cgi-bin/lists?peptidas.txt *********************************************************** * Zinc carboxypeptidases, zinc-binding regions signatures * *********************************************************** There are a number of different types of zinc-dependent carboxypeptidases (EC 3.4.17.-) [1,2]. All these enzymes seem to be structurally and functionally related. The enzymes that belong to this family are listed below. Carboxypeptidase Al (EC 3.4.17.1), a pancreatic digestive enzyme that can removes all C-terminal amino acids with the exception of Arg, Lys and Pro. Carboxypeptidase A2 (EC 3.4.17.15), a pancreatic dd digestive enzyme with a specificity similar to that of carboxypeptidase AI, but with a preference for bulkier C-terminal residues. Carboxypeptidase B (EC 3.4.17.2), also a pancreatic digestive enzyme, but that preferentially removes C-terminal Arg and Lys. Carboxypeptidase N (EC 3.4.17.3) (also known as arginine carboxypeptidase), a plasma enzyme which protects the body from potent vasoactive and inflammatory peptides containing C-terminal Arg or Lys (such as kinins or anaphylatoxins) which are released into the circulation. Carboxypeptidase H (EC 3.4.17.10) (also known as enkephalin convertase or carboxypeptidase E), an enzyme located in secretory granules ofpancreatic islets, adrenal gland, pituitary and brain. This enzyme removes residual C-terminal Arg or Lys remaining after initial endoprotease cleavage during prohormone processing. Carboxypeptidase M (EC 3.4.17.12), a membrane bound Arg and Lys specific enzyme. It is ideally situated to act on peptide hormones at local tissue sites where it could control their activity before or after interaction with specific plasma membrane receptors. Mast cell carboxypeptidase (EC 3.4.17.1), an enzyme with a specificity to carboxypeptidase A, but found in the secretory granules of mast cells. Streptomyces griseus carboxypeptidase (Cpase SG) (EC 3.4.17.-) [3], which combines the specificities of mammalian carboxypeptidases A and B. Thermoactinomyces vulgaris carboxypeptidase T (EC 3.4.17.18) (CPT) [4], which also combines the specificities of carboxypeptidases A and B. AEBPI [5], a transcriptional repressor active in preadipocytes. AEBPI seems to regulate transcription by cleavage of other transcriptional proteins. Yeast hypothetical protein YHRI32c. All of these enzymes bind an atom of zinc. Three conserved residues are implicated in the binding of the zinc atom: two histidines and a glutamic acid. We have derived two signature patterns which contain these three zinc-ligands. -Consensus pattern: [PK]-x-[LIVMFY]-x-[LIVMFY]-x(4)-H-[STAG]-x-E-x-[LIVM]- [STAG]-x(6)-[LIVMFYTA] [H and E are zinc ligands] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: Bacillus sphaericus endopeptidase I which hydrolyses the gamma-D-Glu-(L)meso-diaminopimelic acid bond of spore cortex peptidoglycan [6] and which is possibly distantly related to zinc carboxypeptidases. -Consensus pattern: H-[STAG]-x(3)-[LIVME]-x(2)-[LIVMFYW]-P-[FYW] [H is a zinc ligand] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 52. -Note: if a protein includes both signatures, the probability ofit being a eukaryotic zinc carboxypeptidase is 100% -Note: these proteins belong to families M14A/MI4B in the classification ofpeptidases [7,El]. -Last update: November 1995/ Patterns and text revised. [ 1] Tan F., Chan S.J., Steiner D.F., Schilling J.W., Skidgel R.A. J. BioI. Chern. 264:13165-13170(1989). [ 2] Reynolds D.S., Stevens R.L., Gurley D.S., Lane W.S., Austen K.F., Serafin W.E. J. BioI. Chern. 264:20094-20099(1989). [3] Narahashi Y. J. Biochem. 107:879-886(1990). ee [ 4] Teplyakov A., Polyakov K., Obmolova G., Strokopytov B., Kuranova I.,Osterman A.L., Grishin N.V., Smulevitch S.V., Zagnitko O.P., Galperina O.V., Matz M.V., Stepanov V.M. Eur. J. Biochem. 208:281-288(1992). [ 5] He G.-P., Muise A., Li A.W., Ro H.-S. Nature 378:92-96(1995). [ 6] Hourdou M.-L., Guinand M., Vacheron MJ., Michel G., Denoroy L., Duez C.M., Englebert S., Joris B., Weber G., Ghuysen 1.-M. Biochem.1. 292:563-570(1993). [7] Rawlings N.D., Barrett A.J. Meth. Enzymol. 248:183-228(1995). [E 1] http://www.expasy.org/cgi-binllists?peptidas.txt *************************************************** * ATP-dependent DNA ligase signatures and profile * *************************************************** DNA ligase (polydeoxyribonucleotide synthase) is the enzyme that joins two DNA fragments by catalyzing the formation of an internucleotide ester bond between phosphate and deoxyribose. It is active during DNA replication, DNA repair and DNA recombination. There are two forms of DNA ligase: one requires ATP (EC 6.5.1.1), the other NAD (EC 6.5.1.2). Eukaryotic, archaebacterial, virus and phage DNA ligases are ATP-dependent. During the first step of the joining reaction, the ligase interacts with ATP to form a covalent enzyme-adenylate intermediate. A conserved lysine residue is the site of adenylation [1,2]. Apart from the active site region, the only conserved region common to all ATP-dependent DNA ligases is found [3] in the C-terminal section and contains a conserved glutamate as well as four positions with conserved basic residues. We developed signature patterns for both conserved regions. -Consensus pattern: [EDQH]-x-K-x-[DN]-G-x-R-[GACIVM][K is the active site residue] Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 33. -Consensus pattern: E-G-[LIVMA]-[LIVM](2)-[KR]-x(5,8)-[YW]-[QNEK]-x(2,6) [KRH]-x(3,5)-K-[LIVMFY]-K -Sequences known to belong to this class detected by the pattern: ALL, except for archebacterial DNA ligases. Other sequence(s) detected in Swiss-Prot: NONE. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: 1. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: December 2001/ Text revised; profile added. [ 1] Tomkinson A.E., Totty N.F., Ginsburg M., Lindahl T. PNAS 88:400-404(1991). [2] Lindahl T., Bames D.E. Annu. Rev. Biochem. 61:251-281(1992). [3] Kletzin A. Nucleic Acids Res. 20:5389-5396(1992). ************************************************************** * G-protein coupled receptors family 1 signature and profile * ************************************************************** G-protein coupled receptors [1 to 4,El,E2] (also called R7G) are an extensive group of hormones, neurotransmitters, odorants and light receptors which transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins. The receptors that are currently known to belong to this family are listed below. ff - 5-hydroxytryptamine (serotonin) lA to IF, 2A to 2C, 4, 5A, 5B, 6 and 7 [5]. Acetylcholine, muscarinic-type, Ml to M5. Adenosine AI, A2A, A2B and A3 [6]. Adrenergic alpha-lA to -lC; alpha-2A to -2D; beta-l to -3 [7]. Angiotensin II types I and II. Bombesin subtypes 3 and 4. Bradykinin Bland B2. c3a and C5a anaphylatoxin. Cannabinoid CBl and CB2. Chemokines C-C CC-CKR-l to CC-CKR-8. Chemokines C X-C CXC-CKR-l to CXC-CKR-4. Cholecystokinin-A and cholecystokinin-B/gastrin. Dopamine Dl to D5 [8]. Endothe1in ET-a and ET-b [9]. fMet-Leu-Phe (fMLP) (N-formyl peptide). Follicle stimulating hormone (FSH-R) [10]. Galanin. Gastrin-releasing peptide (GRP-R). Gonadotropin-releasing hormone (GNRH-R). Histamine HI and H2 (gastric receptor I). Lutropin-choriogonadotropic hormone (LSH-R) [10]. Melanocortin MClR to MC5R. Melatonin. Neuromedin B (NMB-R). Neuromedin K (NK-3R). Neuropeptide Y types 1 to 6. Neurotensin (NT-R). Octopamine (tyramine), from insects. Odorants [11]. Opioids delta-, kappa- and mu-types [12]. Oxytocin (OT-R). Platelet activating factor (PAF-R). Prostacyclin. Prostaglandin D2. Prostaglandin E2, EPI to EP4 subtypes. Prostaglandin F2. Purinoreceptors (ATP) [13]. Somatostatin types 1 to 5. Substance-K (NK-2R). Substance-P (NK-lR). Thrombin. Thromboxane A2. Thyrotropin (TSH-R) [10]. Thyrotropin releasing factor (TRH-R). Vasopressin Via, V1b and V2. Visual pigments (opsins and rhodopsin) [14]. Proto-oncogene mas. A number of orphan receptors (whose ligand is not known) from mammals and birds. Caenorhabditis elegans putative receptors C06G4.5, C38C10.l, C43C3.2, T27D1.3 and ZC84.4. Three putative receptors encoded in the genome ofcytomegalovirus: US27, US28, and UL33. ECRF3, a putative receptor encoded in the genome ofherpesvirus saimiri. The structure of all these receptors is thought to be identical. They have seven hydrophobic regions, each of which most probably spans the membrane. The N terminus is located on the extracellular side ofthe membrane and is often glycosylated, while the C-terminus is cytoplasmic and generally phosphorylated. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. Most, but not all ofthese receptors, lack a signal peptide. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops. A conserved acidic-Arg-aromatic triplet is present in the N-terminal extremity of the second cytoplasmic loop [15] and could be implicated in the interaction with G proteins. To detect this widespread family of proteins we have developed a pattern that contains the conserved triplet and that also spans the major part ofthe third transmembrane helix. We have also developed a profile that spans the seven transmembrane regions. -Consensus pattern: [GSTALIVMFYWC]-[GSTANCPDE]- {EDPKRH}-x(2) LIVMNQGA]-x(2)-[LIVMFT]-[GSTANC]-[LIVMFYWSTAC]-[DENH]-R [FYWCSH]-x(2)-[LIVM] -Sequences known to belong to this class detected by the pattern: the majority of receptors. About 5% are not detected. Other sequence(s) detected in Swiss-Prot: 64. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: 1. gg -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Expert(s) to contact by email: Attwood T.K.; [email protected] Kolakowski L.F. Jr.; [email protected] -Last update: December 2001/ Text revised; profile added. [ 1] Strosberg A.D. Eur. J. Biochem. 196:1-10(1991). [2] Kerlavage A.R. Curr. Opin. Struct. BioI. 1:394-401(1991). [ 3] Probst W.C., Snyder L.A., Schuster D.I., Brosius J., Sealfon S.C. DNA Cell BioI. 11:1-20(1992). [4] Savarese T.M., Fraser C.M. Biochem.1. 283:1-9(1992). [5] Branchek T. Curr. BioI. 3:315-317(1993). [ 6] Stiles G.L. J. BioI. Chern. 267:6451-6454(1992). [ 7] Friell T., Kobilka B.K., Lefkowitz R.J., Caron M.G. Trends Neurosci. 11:321 324(1988). [8] Stevens C.F. Curro BioI. 1:20-22(1991). [ 9] Sakurai T., Yanagisawa M., Masaki T. Trends Pharmacoi. Sci. 13:103-107(1992). [10] Salesse R., Remy J.J., Levin J.M., Jallal B., Gamier J. Biochimie 73:109- 120(1991). [11] Lancet D., Ben-Arie N. Curro BioI. 3:668-674(1993). [12] Uhl G.R., Childers S., Pasternak G. Trends Neurosci. 17:89-93(1994). [13] Barnard E.A., Burnstock G., Webb T.E. Trends Pharmacoi. Sci. 15:67-70(1994). [14] Applebury M.L., Hargrave P.A. Vision Res. 26:1881-1895(1986). [15] Attwood T.K., Eliopoulos E.E., Findlay J.B.C. Gene 98:153-159(1991). [El] http://www.gcrdb.uthscsa.edu/ [E2] http://swift.embl-heidelberg.de/7tm/ ************************************************** * Trp-Asp (WD-40) repeats signature and profiles * ************************************************** Beta-transducin (G-beta) is one of the three subunits (alpha, beta, and gamma) of the guanine nucleotide-binding proteins (G proteins) which act as intermediaries in the transduction of signals generated by transmembrane receptors [1]. The alpha subunit binds to and hydrolyzes GTP; the functions ofthe beta and gamma subunits are less clear but they seem to be required for the replacement of GDP by GTP as well as for membrane anchoring and receptor recognition. In higher eukaryotes G-beta exists as a small multigene family of highly conserved proteins of about 340 amino acid residues. Structurally G-beta consists of eight tandem repeats of about 40 residues, each containing a central Trp-Asp motif (this type of repeat is sometimes called a WD-40 repeat). Such a repetitive segment has been shown [E1,2,3,4,5] to exist in a number ofother proteins listed below: - Yeast STE4, a component ofthe pheromone response pathway. STE4 is a G-beta like protein that associates with GPAI (G-alpha) and STE18 (G-gamma). Yeast MSIl, a negative regulator of RAS-mediated cAMP synthesis. MSIl is most probably also a G-beta protein. Human and chicken protein 12.3. The function of this protein is not hh known, but on the basis of its similarity to G-beta proteins, it may also function in signal transduction. Chlamydomonas reinhardtii gblp. This protein is most probably the homolog of vertebrate protein 12.3. Human LIS1, a neuronal protein involved in type-l lissencephaly [E2]. Mammalian coatomer beta' subunit (beta'-COP), a component of a cytosolic protein complex that reversibly associates with Golgi membranes to form vesicles that mediate biosynthetic protein transport. Yeast CDC4, essential for initiation of DNA replication and separation of the spindle pole bodies to form the poles of the mitotic spindle. Yeast CDC20, a protein required for two microtubule-dependent processes: nuclear movements prior to anaphase and chromosome separation. Yeast MAKll, essential for cell growth and for the replication of Ml double-stranded RNA. Yeast PRP4, a component of the U4/U6 small nuclear ribonucleoprotein with a probable role in mRNA splicing. Yeast PWP1, a protein of unknown function. Yeast SKI8, a protein essential for controlling the propagation of double-stranded RNA. Yeast SOF1, a protein required for ribosomal RNA processing which associates with U3 small nucleolar RNA. Yeast TUP1 (also known as AER2 or SFL2 or CYC9), a protein which has been implicated in dTMP uptake, catabolite repression, mating sterility, and many other phenotypes. Yeast YCR57c, an ORF ofunknown function from chromosome III. Yeast YCR72c, an ORF of unknown function from chromosome III. Slime mold coronin, an actin-binding protein. Slime mold AAC3, a developmentally regulated protein of unknown function. Drosophila protein Groucho (formerly known as E(spl); 'enhancer of split'), a protein involved in neurogenesis and that seems to interact with the Notch and Delta proteins. Drosophila TAF-II-80, a protein that is tightly associated with TFIID. The number ofrepeats in the above proteins varies between 5 (PRP4, TUP1, and Groucho) and 8 (G-beta, STE4, MSIl, AAC3, CDC4, PWP1, etc.). In G-beta and G beta like proteins, the repeats span the entire length of the sequence, while in other proteins, they make up the N-terminal, the central or the C-terminal section. A signature pattern can be developed from the central core ofthe domain (positions 9 to 23). Two profiles were developed for this module, the first one picks up WD repeats while the second profile is 'circular' and will thus detect a region containing adjacent WD repeats. -Consensus pattern: [LIVMSTAC]-[LIVMFYWSTAGC]-[LIMSTAG]-[LIVMSTAGC] x(2)-[DN]-x(2)-[LIVMWSTAC]-x-[LIVMFSTAG]-W-[DEN]-[LIVMFSTAGCN] -Sequences known to belong to this class detected by the pattern: A majority. This pattern does not detect ALL the occurrences ofthe domain in any ofthe above proteins, as some ofthe copies ofthe domain are less conserved. Other sequence(s) detected in Swiss-Prot: 95 other proteins, but in all of them, the pattern is found only ONCE, whereas it is generally found twice or more in WD-repeat proteins. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: 1. Sequences known to belong to this class detected by the circular profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: December 2001 / Text revised; profile added. 11 [ 1] Gilman A.G. Annu. Rev. Biochem. 56:615-649(1987). [2] Duronio R.l, Gordon J.I., Boguski M.S. Proteins 13:41-56(1992). [3] van derVoorn L., Ploegh H.L. FEBS Lett. 307:131-134(1992). [4] Neer E.J., Schmidt C.l, Nambudripad R., Smith T.F. Nature 371:297-300(1994). [ 5] Smith T.F., Gaitatzes C., Saxena K., Neer E.J. Trends Biochem. Sci. 24:181 185(1999). [E 1] http://bmerc-www.bu.edu/wdrepeat/ [E2] http://bioinformatics.weizmann.ac.il/hotmolecbase/entries/lis1.htm **************************************************** * Uncharacterized protein family UPF0057 signature * **************************************************** The following uncharacterized proteins have been shown [1] to be evolutionary related: Barley low-temperature induced protein blt101. Lophorium elongatum salt-sress induced protein ESI3. Yeast hypothetical proteins YDL123w, YDR276c, YDR525Bw and YJL151c. Caenorhabditis elegans hypothetical proteins F47B7.1, T23F2.3, T23F2.4, T23F2.5 and ZK632.10. Escherichia coli hypothetical protein yqaE. Synechocystis strain PCC 6803 hypothetical protein ssrl169. These are small proteins of from 52 to 140 amino-acid resiudes that contains two transmembrane domains. As a signature pattern we selected a region that corresponds to the end ofthe first transmembrane helix. -Consensus pattern: [LIV]-x-[STA]-[LIVF](3)-P-P-[LIVA]-[GA]-[IV]-x(4)-[GKN] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE -Last update: July 1998/ First entry. [ 1] Rudd K.E., Humphery-Smith I., Wasinger V.C., Bairoch A. Electrophoresis 19:536 544(1998). ***************************************************** * Carbamoyl-phosphate synthase subdomain signatures * ***************************************************** Carbamoyl-phosphate synthase (CPSase) catalyzes the ATP-dependent synthesis of carbamyl-phosphate from glutamine (EC 6.3.5.5) or ammonia (EC 6.3.4.16) and bicarbonate [1]. This important enzyme initiates both the urea cycle and the biosynthesis ofarginine and pyrimidines. Glutamine-dependent CPSase (CPSase II) is involved in the biosynthesis of pyrimidines and purines. In bacteria such as Escherichia coli, a single enzyme is involved in both biosynthetic pathways while other bacteria have separate enzymes. The bacterial enzymes are formed oftwo subunits. A small chain (gene carA) that provides glutamine amidotransferase activity (GATase) necessary for removal of the ammonia group from glutamine, and a large chain (gene carB) that provides CPSase activity. Such a structure is also present in fungi for arginine biosynthesis (genes CPA1 and CPA2). In most eukaryotes, the first three steps ofpyrimidine biosynthesis are catalyzed by a large multifunctional enzyme-called URA2 in yeast, rudimentary in Drosophila and CAD in mammals [2]. The CPSase domain is located between an N-terminal GATase domain and the C-terminal part which encompass the dihydroorotase and aspartate transcarbamylase activities. JJ Ammonia-dependent CPSase (CPSase I) is involved in the urea cycle in ureolytic vertebrates; it is a monofunctional protein located in the mitochondrial matrix. The CPSase domain is typically 120 Kd in size and has arisen from the duplication of an ancestral subdomain of about 500 amino acids. Each subdomain independently binds to ATP and it is suggested that the two homologous halves act separately, one to catalyze the phosphorylation of bicarbonate to carboxy phosphate and the other that of carbamate to carbamyl phosphate. The CPSase subdomain is also present in a single copy in the biotin-dependent enzymes acetyl-CoA carboxylase (EC 6.4.1.2) (ACC), propionyl-CoA carboxylase (EC 6.4.1.3) (PCCase), pyruvate carboxylase (EC 6.4.1.1) (PC) and urea carboxylase (EC 6.3.4.6). As signatures for the subdomain, we selected two conserved regions which are probably important for binding ATP and/or catalytic activity. -Consensus pattern: [FYV]-[PS]-[LIVMC]-[LIVMA]-[LIVM]-[KR]-[PSA]-[STA]-x(3) [SG]-G-x-[AG] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Consensus pattern: [LIVMF]-[LIMN]-E-[LIVMCA]-N-[PATLIVM]-[KR] [LIVMSTAC] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 35. -Last update: July 1998/ Patterns and text revised. [ 1] Simmer J.P., Kelly R.E., Rinker A.G. Jr., Scully J.L., Evans D.R. J. BioI. Chern. 265: 10395-10402(1990). [ 2] Davidson J.N., Chen K.C., Jamison R.S., Musmanno L.A., Kern C.B. BioEssays 15: 157-164(1993). *********************************** * Ribosomal protein L29 signature * *********************************** Ribosomal protein L29 is one of the proteins from the large ribosomal subunit. L29 belongs to a family ofribosomal proteins which, on the basis ofsequence similarities [1]. ], groups: Eubacterial L29. Red algal L29. Archaebacterial L29. Mammalian L35. Caenorhabditis elegans L35 (ZK652.4). Yeast L35. L29 is a protein of 63 to 138 amino acid residues. As a signature pattern, we selected a conserved region located in the central section ofL29. -Consensus pattern:[KNQS]-[PSTLNH]-x(2)-[LIVMFA]-[KRGSADN]-x-[LIVYSTA] [KR]- [KRHQS]-[DESTANQRL]-[LIV]-A-[KRCQVT]-[LIVMA] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 2. -Last update: December 2001 / Pattern and text revised. [ 1] Otaka E., Hashimoto T., Mizuta K. Protein Seq. Data Anal. 5:285-300(1993). ******************************************************* * Heavy-metal-associated domain signature and profile * •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• A conserved domain of about 70 amino acid residues has been found [1] in a number of proteins that transport or detoxify heavy metals. This domain contains two conserved cysteines that could be involved in the binding of these metals. The domain has been kk termed Heavy-Metal-Associated (HMA). Solution structure of the fourth HMA domain of the Menkes copper-transporting ATPase shows a well defined structure comprising a four-stranded antiparalle1 beta-sheet and two alpha helices packed in an alpha-beta sandwich fold (see ) [2]. This fold is common to other domains and is classified as "ferredoxin-like". Some ofthe proteins containing an HMA domain are listed below. -A variety of cation transport ATPases (EI-E2 ATPases) (see The profile we developed spans the complete domain. The pattern is centered on the two metal-binding residues. -Consensus pattern: [LIVNS]-x(2)-[LIVMFA]-x-C-x-[STAGCDNH]-C-x(3)-[LIVFG]- x(3)-[LIV]-x(9,11)-[IVA]-x-[LVFYS] [The 2 C's may bind metals] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 7. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: May 2004 / Text revised. [ 1] Bull P.C., Cox D.W. Trends Genet. 10:246-252(1994). [ 2] Gitschier J., Moffat B., Reilly D., Wood W.I., Fairbrother W.J. Nat. Struct. BioI. 5:47-54(1998). [ 3] Lin S.-J., Culotta V.L. Proc. Natl. Acad. Sci. U.S.A. 92:3784-3788(1995). 11 ********************************************************************* * ATP-binding cassette, ABC transporter-type, signature and profile * ********************************************************************* ABC transporters belong to the ATP-Binding Cassette (ABC) superfamily which uses the hydrolysis of ATP to energize diverse biological systems. ABC transporters are minimally constituted of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These regions can be found on the same protein or on two different ones. Most ABC transporters function as a dimer and therefore are constituted of four domains, two ABC modules and two TMDs [1]. ABC transporters are involved in the export or import of a wide variety of substrates ranging from small ions to macromolecules. The major function of ABC import systems is to provide essential nutrients to bacteria. They are found only in prokaryotes and their four constitutive domains are usually encoded by independent polypeptides (two ABC proteins and two TMD proteins). Prokaryotic importers require additional extracytoplasmic binding proteins (one or more per systems) for function. In contrast, export systems are involved in the extrusion of noxious substances, the export of extracellular toxins and the targeting of membrane components. They are found in all living organisms and in general the TMD is fused to the ABC module in a variety of combinations. Some eukaryotic exporters encode the four domains on the same polypeptide chain [2,3]. The ABC module (approximately two hundred amino acid residues) is known to bind and hydrolyze ATP, thereby coupling transport to ATP hydrolysis in a large number of biological processes. The cassette is duplicated in several subfamilies. Its primary sequence is highly conserved, displaying a typical phosphate-binding loop: Walker A (see As a signature pattern for this class of proteins, we use a conserved region which is located between the 'A' and the 'B' motifs of the ATP-binding site. The profile we developed is directed against the conserved ABC module by covering the region between beta strand 1 and alpha helix 9, including not only the conserved motifs but also structural elements found Nand C terminal to them. Our profile also recognizes the UvrA family which is evolutionarily related to the ABC transporter family. -Consensus pattern: [LIVMFYC]-[SA]-[SAPGLVFYKQH]-G-[DENQMW] [KRQASPCLIMFW]-[KRNQSTAVM]~[KRACLVM]-[LIVMFYPAN]- {PHY} [LIVMFW]-[SAGCLIVP]-{FYWHP}-{KRHP}-[LIVMFYWSTA] nn -Sequences known to belong to this class detected by the pattern: ALL, except for 25 sequences. Other sequence(s) detected in Swiss-Prot: 53. Sequences known to belong to this class detected by the profile: ALL. Other sequence(s) detected in Swiss-Prot: NONE. -Note: the ATP-binding region is duplicated in araG, mdl, msrA, rbsA, tlrC, uvrA, yejF, Mdr's, CFTR, pmdl and in EF-3. In some ofthose proteins, the above pattern only detect one ofthe two copies ofthe domain. -Note: this documentation entry is linked to both a signature pattern and a profile. As the profile is much more sensitive than the pattern, you should use it ifyou have access to the necessary software tools to do so. -Last update: November 2003/ Text revised. [ 1] Holland LB., Cole S.P.C., Kuchler K., Higgins C.F. (In) ABC proteins from bacteria to man, Academic Press, San Diego, (2003). [2] Holland I.B., Blight M.A. J. Mol. BioI. 293:381-399(1999). [ 3] Saurin W., Hofnung M., Dassa E. 1. Mol. Evol. 48:22-41(1999). [ 4] Higgins C.F. Res. Microbiol. 152:205-210(2001). [ 5] Higgins C.F. Annu. Rev. Cell. BioI. 8:67-113(1992). [ 6] Schneider E., Hunke S. FEMS Microbiol. Rev. 22:1-20(1998). [7] Kerr LD. Biochim. Biophys. Acta 1561:47-64(2002). [ 8] Karpowich N., Martsinkevich 0., Millen L., Yuan Y.R., Dai P.L., MacVey K., Thomas PJ., Hunt J.F. Structure 9:571-586(2001). [ 9] Yuan YR., Blecker S., Martsinkevich 0., Millen L., Thomas P.J., Hunt J.F. J. BioI. Chern. 276:32313-32321(2001). [10] Hung L.W., Wang LX., Nikaido K., Liu P.Q., Ames G.F., Kim S.H. Nature 396:703 707(1998). [11] Diederichs K., Diez J., Greller G., Muller c., Breed J., Schnell C., Vonrhein C., Boos W., Welte W. EMBO J. 19:5951-5961(2000). [12] Gaudet R., Wiley D.C. EMBO J. 20:4964-4972(2001). [E1] http://tcdb.ucsd.edu/tcdb/tcfamilybrowse.php?tcname=3.A.l ********************* * Serpins signature * ********************* Serpins (SERine Proteinase INhibitors) [1,2,3,4] are a group of structurally related proteins. They are high molecular weight (400 to 500 amino acids), extracellular, irreversible serine protease inhibitors with a well defined structural-functional characteristic: a reactive region that acts as a 'bait' for an appropriate serine protease. This region is found in the C-terminal part ofthese proteins. Proteins which are known to belong to the serpin family are listed below (references are only provided for recently determined sequences): Alpha-l protease inhibitor (alpha-I-antitrypsin, contrapsin). Alpha-l-antichymotrypsin, Antithrombin III. Alpha-2-antiplasmin. Heparin cofactor II. Complement Cl inhibitor. Plasminogen activator inhibitors 1 (PAl-I) and 2 (PAI-2). Glia derived nexin (GDN) (Protease nexin I). Protein C inhibitor. Rat hepatocytes SPI-l, SPI 2 and SPI-3 inhibitors. Human squamous cell carcinoma antigen (SCCA) which may act in the modulation of the host immune response against tumor cells. A lepidopteran protease inhibitor. Leukocyte elastase inhibitor which, in contrast to other serpins, is an intracellular protein. Neuroserpin [5], a neuronal inhibitor of plasminogen activators 00 and plasmin. Cowpox virus crmA [6], an inhibitor ofthe thiol protease interleukin-IB converting enzyme (ICE). CrmA is the only serpin known to inhibit a non-serine proteinase. Some orthopoxviruses probable protease inhibitors, which may be involved in the regulation of the blood clotting cascade and/or ofthe complement cascade in the mammalian host. On the basis of strong sequence similarities, a number of proteins with no known inhibitory activity are said to belong to this family: Birds ovalbumin and the related genes X and Y proteins. Angiotensinogen; the precursor of the angiotensin active peptide. Barley protein Z; the major endosperm albumin. Corticosteroid binding globulin (CBG). Thyroxine-binding globulin (TBG). Sheep uterine milk protein (UTMP) and pig uteroferrin-associated protein (UFAP). Hsp47, an endoplasmic reticulum heat-shock protein that binds strongly to collagen and could act as a chaperone in the collagen biosynthetic pathway [7]. Maspin, which seems to function as a tumor supressor [5]. Pigment epithelium-derived factor precursor (PEDF), a protein with a strong neutrophic activity [8]. Ep45, an estrogen-regulated protein from Xenopus [9]. We developed a signature pattern for this family of proteins, centered on a well conserved Pro-Phe sequence which is found ten to fifteen residues on the C-terminal side ofthe reactive bond. -Consensus pattern: [LIVMFY]-x-[LIVMFYAC]-[DNQ]-[RKHQS]-[PST]-F [LIVMFY]-[LIVMFYC]-x-[LIVMFAH] -Sequences known to belong to this class detected by the pattern: ALL, except for 7 sequences. Other sequence(s) detected in Swiss-Prot: 27. -Note: in position 6 ofthe pattern, Pro is found in most serpins. -Last update: July 1998 I Text revised. [ 1] Carrell R, Travis J. Trends Biochem. Sci. 10:20-24(1985). [ 2] Carrell R., Pemberton P.A., Boswell D.R. Cold Spring Harbor Symp. Quant. BioI. 52:527-535(1987). [3] Huber R, Carrell R.W. Biochemistry 28:8951-8966(1989). [4] Remold-O'Donneel E. FEBS Lett. 315:105-108(1993). [ 5] Osterwalder T., Contartese J., Stoeckli E.T., Kuhn T.B., Sonderegger P. EMBO J. 15:2944-2953(1996). [6] Komiyama T., Ray C.A., Pickup D.l, Howard AD., Thornberry N.A, Peterson E.P., Salvesen G. J. BioI. Chern. 269:19331-19337(1994). [7] Clarke E., Sandwal RD. Biochim. Biophys. Acta 1129:246-248(1992). [ 8] Zou Z., Anisowicz A, Neveu M., Rafidi K., Sheng S., Sager R, Hendrix M.J., Seftor E., Thor A Science 263:526-529(1994). [ 9] Steele F.R., Chader G.J., Johnson L.V., Tombran-Tink J. PNAS 90:1526- 1530(1993). [10] Holland L.l, Suksang C., Wall AA., Roberts L.R., Moser D.R., Bhattacharya A. 1 BioI. Chern. 267:7053-7059(1992). pp ******************************************* * Cysteine proteases inhibitors signature * ******************************************* Inhibitors ofcysteine proteases [1,2,3], which are found in the tissues and body fluids of animals, in the larva of the worm Onchocerca volvulus [4], as well as in plants, can be grouped into three distinct but related families: - Type 1 cystatins (or stefins), molecules of about 100 amino acid residues with neither disulfide bonds nor carbohydrate groups. - Type 2 cystatins, molecules ofabout 115 amino acid residues which contain one or two disulfide loops near their C-terminus. - Kininogens, which are multifunctional plasma glycoproteins. They are the precursor of the active peptide bradykinin and playa role in blood coagulation by helping to position optimally prekallikrein and factor XI next to factor XII. They are also inhibitors of cysteine proteases. Structurally, kininogens are made of three contiguous type-2 cystatin domains, followed by an additional domain (of variable length) which contains the sequence of bradykinin. The first of the three cystatin domains seems to have lost its inhibitory activity. In all these inhibitors, there is a conserved region of five residues which has been proposed to be important for the binding to the cysteine proteases. Our pattern starts one residue before this conserved region. -Consensus pattern: [GSTEQKRV]-Q-[LIVT]-[VAF]-[SAGQ]-G-x-[LIVMNK]-x(2)- [LIVMFY]-x-[LIVMFYA]-[DENQKRHSIV] -Sequences known to belong to this class detected by the pattern: ALL. Other sequence(s) detected in Swiss-Prot: 11. -Note: this pattern is always twice in kininogens. -Note: members of the fetuin family (see PROSITE result file (text): [PROSITE] User: [email protected]. Last modification time: Sat Nov 13 10:21:01 2004. Current time: Sat Nov 13 10:21:01 2004 This service is supported by Ministere de la recherche (ACC-SV13), CNRS (IMABIO, COMI, GENOME) and Region Rhone-Alpes (Programme EMERGENCE) .. PROSITE result file (text): [PROSITE] User: [email protected]. Last modification time: Sun Nov 14 05:33:09 2004. Current time: Sun Nov 14 05:33:09 2004 This service is supported by Ministere de la recherche (ACC-SV13), CNRS (IMABIO, COMI, GENOME) and Region Rhone-Alpes (Programme EMERGENCE) .