DOCSLIB.ORG

Mechanisms of Presynaptic Cav2.2 (N-Type) Modulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanisms of Presynaptic Cav2.2 (N-Type) Modulation Mechanisms of presynaptic CaV2.2 (N-type) modulation by Allen W. Chan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Physiology University of Toronto ©Copyright by Allen Chan 2008 ii Abstract Mechanisms of presynaptic CaV2.2 (N-type) modulation. Allen W. Chan Doctor of Philosophy, 2008 Department of Physiology University of Toronto Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPγS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. iii In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation. iv Acknowledgements I would like to extend my deepest gratitude to my supervisor, Dr. Elise Stanley. From the moment I began my studies, she provided an intense intellectually stimulating and challenging environment. She has taught me to be rigorous in all manners of scientific thought, to be mindful of the nuances of experimental limitations, and yet to also remain open-minded to the often unexpected possibilities that lay in science. Above and beyond her duties as a supervisor, I would like to also extend my sincerest gratitude to Dr. Stanley for her advice, her sympathy and her support during a time when a severe illness in my family threatened to derail my studies and my world as I knew it. What was unquestionably the darkest time in my life was made lighter with her help. I would also like to extend my appreciation to the members of my supervisory committee, Dr. Lyanne Schlichter and Dr. Frances Skinner. Their advice and encouragement throughout my studies have been invaluable and truly appreciated. Also, thank you to the members of my doctoral transfer committee, Dr. Harold Atwood, Dr. Jane Mitchell, and Dr. Shuzo Sugita. A special thanks to Dr. Qi Li, who has always been willing to help and provide technical support for almost every facet of my work in the laboratory. Thank you to Dr. Steven Owens, Dr. Evan Newell and Luigi Gentile for their advice, tutelage, and camaraderie in all aspects of the intricacies and frustrations of electrophysiology. Thank you to current v and past members of the laboratory, Dr. Rajesh Khanna, Dr. Terence Morris, Fiona Wong, and Sabiha Gardezi for the incalculable support, discussion, and friendship. Thank you to my parents who have supported me throughout these years and trusted that what I was doing was important. For financial support, I have been the beneficiary of fellowships from the University of Toronto. Also, a special thank you to the Ontario Student Opportunities Trust Fund (OSOTF) for awarding me the Margaret and Howard Gamble Research Grant, and to the Canadian Institutes of Health Research (CIHR) for their generous support in awarding me the doctoral Sir Frederick Banting and Dr. Charles Best Canada Graduate Scholarship. Finally, a special thank you to Erika without whose unconditional support, encouragement, and optimism, none of this would be possible. Her spirit has, and will always remain, an inspiration for me to do the best I can in life. vi Table of Contents Abstract ......................................................................................................................................ii Acknowledgements .................................................................................................................. iv Table of Contents ...................................................................................................................... vi List of Figures ............................................................................................................................ ix Abbreviations ............................................................................................................................ xi Chapter 1 : Introduction .......................................................................................................... 1 1.1 Introduction to presynaptic calcium channels ............................................................................ 1 1.2 Molecular structure of voltage‐gated calcium channels ............................................................. 3 1.2.1 Calcium channel α1 subunit ................................................................................................. 3 1.2.2 Calcium channel auxiliary subunits ...................................................................................... 8 1.3 G‐protein modulation of calcium channels ............................................................................... 11 1.3.1 G‐protein coupled receptor signaling ................................................................................ 11 1.3.2 Voltage‐dependent G‐protein modulation ........................................................................ 13 1.3.3 G‐protein interaction with syntaxin ................................................................................... 18 1.3.4 Voltage‐independent G‐protein modulation ..................................................................... 19 1.3.4.1 Voltage‐independent G‐protein modulation via tyrosine kinase signaling ................... 19 1.3.4.2 Voltage‐independent G‐protein modulation via PIP2 regulation .................................... 20 1.4 PKC phosphorylation ................................................................................................................. 22 1.4.1 Crosstalk between PKC & G‐protein signaling pathways ................................................... 22 1.5 Calcium channel regulation by synaptic proteins ..................................................................... 24 1.5.1 Syntaxin‐1/Munc‐18 ........................................................................................................... 24 1.6 Experimental models ................................................................................................................. 26 1.6.1 Chick ciliary ganglion calyx synapse ................................................................................... 26 1.6.2 Chick dorsal root ganglion neurons .................................................................................... 30 Chapter 2 : Materials and methods ....................................................................................... 32 2.1 Preparation of the ciliary ganglion synapse .............................................................................. 32 2.2 Preparation of dorsal root ganglion (DRG) neurons ................................................................. 33 vii 2.3 Patch‐clamp recording .............................................................................................................. 34 2.4 Drug treatments ........................................................................................................................ 35 2.5 DRG transfection ....................................................................................................................... 36 2.6 Statistical analysis ...................................................................................................................... 37 Chapter 3 : Kinetic analysis of G‐protein inhibition of CaV2.2 by G‐protein coupled receptor activation and direct G‐protein activation with GTPγS .......................................................... 38 3.1 Summary ................................................................................................................................... 38 3.2 Introduction ..............................................................................................................................
Load more

Recommended publications
  • Facilitation of Rabbit ב1B Calcium Channels: Involvement of Endogenous Gגד Subunits
    Keywords: Calcium channel, G protein, Facilitation 7535 Journal of Physiology (1998), 509.1, pp.15—27 15 Facilitation of rabbit á1B calcium channels: involvement of endogenous Gâã subunits Gary J. Stephens, Nicola L. Brice, Nicholas S. Berrow and Annette C. Dolphin Department of Pharmacology, University College London and Royal Free Hospital School of Medicine, London WC1E 6BT, UK (Received 30 October 1997; accepted after revision 26 January 1998) 1. The á1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gâã subunits. Using transient expression in COS_7 cells of á1B together with the accessory subunits áµ—ä and â2a,wehaveexaminedtheroleofendogenous Gâã subunits in the tonic modulation of á1B, and compared this with modulation by exogenously expressed Gâã subunits. 2. Prepulse facilitation of control á1BÏáµ—äÏâ2a currents was always observed. This suggests the existenceoftonicmodulationofá1B subunits. To determine whether endogenous Gâã is involved in the facilitation observed in control conditions, the âARK1 Gâã-binding domain (amino acids 495—689) was overexpressed, in order to bind free Gâã subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPâS also reduced the control facilitation. 3. Co-expression of the GâÔãµ subunit, together with the á1BÏáµ—äÏâ2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation.
    [Show full text]
  • An Unexpected Role for Brain-Type Sodium Channels in Coupling of Cell Surface Depolarization to Contraction in the Heart
    An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart Sebastian K. G. Maier*, Ruth E. Westenbroek*, Kenneth A. Schenkman†, Eric O. Feigl‡, Todd Scheuer*, and William A. Catterall*§ Departments of *Pharmacology, †Pediatrics, and ‡Physiology and Biophysics, University of Washington, Seattle, WA 98195 Contributed by William A. Catterall, December 27, 2001 ␣ Voltage-gated sodium channels composed of pore-forming and ventricular myocytes compared with Nav1.5, but they contribute auxiliary ␤ subunits are responsible for the rising phase of the significantly to coupling of cell surface depolarization to con- action potential in cardiac muscle, but the functional roles of traction because of their location in the transverse tubules. Our distinct sodium channel subtypes have not been clearly defined. results provide the first evidence, to our knowledge, for a unique Immunocytochemical studies show that the principal cardiac pore- functional role of TTX-sensitive brain-type sodium channels in forming ␣ subunit isoform Nav1.5 is preferentially localized in the heart. intercalated disks, whereas the brain ␣ subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium Methods currents due to the highly tetrodotoxin (TTX)-sensitive brain iso- All procedures were approved by the Institutional Animal Care forms in the transverse tubules are small and are detectable only and Use Committee of the University of Washington. after activation with ␤ scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface mem- Cell Isolation. Ventricular myocytes were isolated from adult male brane to contraction, because low TTX concentrations reduce left (8–10 weeks) wild-type B6129F1 mice, as described (14).
    [Show full text]
  • The Role of S4 Charges in Voltage-Dependent
    ARTICLE The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes Gianina Panaghie and Geoffrey W. Abbott Greenberg Division of Cardiology, Department of Medicine and Department of Pharmacology, Cornell University, Weill Medical College, New York, NY 10021 Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage- dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments re- vealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitu- tive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels.
    [Show full text]
  • ACTX-Hv1c, Isolated from the Venom of the Blue Mountains
    S.J. Gunning et al. Janus-faced atracotoxins block KCa channels The Janus-faced atracotoxins are specific blockers of invertebrate KCa channels Simon J. Gunning1, Francesco J. Maggio2,4, Monique J. Windley1, Stella M. Valenzuela1, Glenn F. King3 and Graham M. Nicholson1 1 Neurotoxin Research Group, Department of Medical & Molecular Biosciences, University of Technology, Sydney, Broadway, NSW 2007, Australia 2 Department of Molecular, Microbial & Structural Biology, University of Connecticut School of Medicine, Farmington, Connecticut 06032, USA and 3 Division of Chemical and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia 4 Current affiliation: Bristol-Myers Squibb, 6000 Thompson Road, Syracuse NY 13057, USA Subdivision: Molecular Neurobiology Abbreviations: ACTX, atracotoxin; 4-AP, 4-aminopyridine; BKCa channel, 2+ + 2+ large-conductance Ca -activated K channel; CaV channel, voltage-activated Ca channel; ChTx, charybdotoxin; DRG, dorsal root ganglia; DUM, dorsal unpaired median; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N’N’-tetraacetic acid; IbTx, + iberiotoxin; J-ACTX, Janus-faced atracotoxin; KA channel, transient ‘A-type’ K channel; 2+ + + KCa channel, Ca -activated K channel; KDR channel, delayed-rectifier K channel; KV + + channel, voltage-activated K channel; NaV channel, voltage-activated Na channel; Slo, Slowpoke; TEA, tetraethylammonium; TTX, tetrodotoxin. RUNNING TITLE: Janus-faced atracotoxins block KCa channels ADDRESS CORRESPONDENCE TO: Graham M. Nicholson, Department of Medical & Molecular Biosciences, University of Technology, Sydney, PO Box 123, Broadway NSW 2007, Australia. Tel: +61 2 9514-2230 Fax: +61 2 9514-2228; E-mail: [email protected] 1 S.J. Gunning et al. Janus-faced atracotoxins block KCa channels ABSTRACT The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge.
    [Show full text]
  • Uhm Phd 9118021 R.Pdf
    INFORMATION TO USERS The most advanced technology has been used to photograph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. U·M·I University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. M148106·1346 USA 313/761-4700 800/521-0600 Order Number 9118021 Sodium channel activation mechanisms: Insights from deuterium oxide and ddta-9-tetrahydrocannabinol substitution Alicata, Daniel Andrew, Ph.D.
    [Show full text]
  • Phenytoin Inhibits the Persistent Sodium Current in Neocortical Neurons by Modifying Its Inactivation Properties
    Phenytoin Inhibits the Persistent Sodium Current in Neocortical Neurons by Modifying Its Inactivation Properties Elisa Colombo1, Silvana Franceschetti1, Giuliano Avanzini1, Massimo Mantegazza1,2* 1 Department of Neurophysiopathology – Epilepsy Center, Foundation IRCCS Neurological Institute C. Besta, Milan, Italy, 2 Institut de Pharmacologie Mole´culaire et Cellulaire (IPMC), CNRS UMR7275 and University of Nice-Sophia Antipolis, Valbonne, France Abstract + The persistent Na current (INaP) is important for neuronal functions and can play a role in several pathologies, although it is + small compared to the transient Na current (INaT). Notably, INaP is not a real persistent current because it undergoes inactivation with kinetics in the order of tens of seconds, but this property has often been overlooked. Na+ channel blockers, drugs used for treating epilepsy and other diseases, can inhibit INaP, but the mechanism of this action and the conditions in which INaP can be actually inhibited have not been completely clarified yet. We evaluated the action of phenytoin (PHT), a + prototype anti-epileptic Na channel blocker, on INaP inactivation in pyramidal neurons of rat sensorimotor cortical slices at different concentrations, from 5 to 100 mM. PHT did not modify INaP evoked with depolarizing voltage ramps of 50 or 21 21 100 mVs , but decreased INaP evoked by slower voltage ramps (10 mVs ). However, at all of the tested concentrations, PHT decreased INaP evoked by faster ramps when they were preceded by inactivating pre-pulses. Moreover, PHT shifted towards negative potentials the voltage-dependence of INaP inactivation and accelerated its kinetics of development also at depolarized potentials (+40 mV), not consistently with a simple inactivated state stabilizer.
    [Show full text]
  • Bioinsecticides for the Control of Human Disease Vectors Niraj S Bende B
    Bioinsecticides for the control of human disease vectors Niraj S Bende B. Pharm, MRes. Bioinformatics A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience Abstract Many human diseases such as malaria, Chagas disease, chikunguniya and dengue fever are transmitted via insect vectors. Control of human disease vectors is a major worldwide health issue. After decades of persistent use of a limited number of chemical insecticides, vector species have developed resistance to virtually all classes of insecticides. Moreover, considering the hazardous effects of some chemical insecticides to environment and the scarce introduction of new insecticides over the last 20 years, there is an urgent need for the discovery of safe, potent, and eco-friendly bioinsecticides. To this end, the entomopathogenic fungus Metarhizium anisopliae is a promising candidate. For this approach to become viable, however, limitations such as slow onset of death and high cost of currently required spore doses must be addressed. Genetic engineering of Metarhizium to express insecticidal toxins has been shown to increase the potency and decrease the required spore dose. Thus, the primary aim of my thesis was to engineer transgenes encoding highly potent insecticidal spider toxins into Metarhizium in order to enhance its efficacy in controlling vectors of human disease, specifically mosquitoes and triatomine bugs. As a prelude to the genetic engineering studies, I surveyed 14 insecticidal spider venom peptides (ISVPs) in order to compare their potency against key disease vectors (mosquitoes and triatomine bugs) In this thesis, we present the structural and functional analysis of key ISVPs and describe the engineering of Metarhizium strains to express most potent ISVPs.
    [Show full text]
  • Somatic Depolarization Enhances GABA Release in Cerebellar Interneurons Via a Calcium/Protein Kinase C Pathway
    5804 • The Journal of Neuroscience, April 13, 2011 • 31(15):5804–5815 Cellular/Molecular Somatic Depolarization Enhances GABA Release in Cerebellar Interneurons via a Calcium/Protein Kinase C Pathway Brice Bouhours, Federico F. Trigo, and Alain Marty Laboratoire de Physiologie Ce´re´brale, Centre National de la Recherche Scientifique and Universite´ Paris Descartes, 75006 Paris, France In cortical and hippocampal neurons, tonic somatic depolarization is partially transmitted to synaptic terminals, where it enhances transmitter release. It is not known to what extent such “analog signaling” applies to other mammalian neurons, and available evidence concerning underlying mechanisms is fragmentary and partially controversial. In this work, we investigate the presence of analog signaling in molecular layer interneurons of the rat cerebellum. GABA release was estimated by measuring autoreceptor currents in single recordings, or postsynaptic currents in paired recordings of synaptically connected neurons. We find with both assays that moderatesubthresholdsomaticdepolarizationresultsinenhancedGABArelease.Inaddition,changesinthecalciumconcentrationwere investigated in the axon compartment using the calcium-sensitive dye OGB-1 (Oregon Green BAPTA-1). After a step somatic depolariza- tion, the axonal calcium concentration and the GABA release probability rise with a common slow time course. However, the amount of calcium entry that is associated to one action potential is not affected. The slow increase in calcium concentration is inhibited by the P/Q calcium channel blocker ␻-agatoxin-IVA. The protein kinase C inhibitor Ro 31-8220 (3-[3-[2,5-dihydro-4-(1-methyl-1H-indol-3-yl)-2,5- dioxo-1H-pyrrol-3-yl]-1H-indol-1-yl]propyl carbamimidothioic acid ester mesylate) did not affect the calcium concentration changes butitblockedtheincreaseinGABArelease.EGTAwasaweakblockerofanalogsignaling,implicatingacloseassociationofproteinkinase C to the site of calcium entry.
    [Show full text]
  • Mode Shift of the Voltage Sensors in Shaker K+ Channels Is Caused by Energetic Coupling to the Pore Domain
    A r t i c l e Mode shift of the voltage sensors in Shaker K+ channels is caused by energetic coupling to the pore domain Georges A. Haddad1,2 and Rikard Blunck1,2,3 1Groupe d’étude des protéines membranaires, 2Département de physique, and 3Département de physiologie, Université de Montréal, Montréal, Québec H3C 3J7, Canada The voltage sensors of voltage-gated ion channels undergo a conformational change upon depolarization of the membrane that leads to pore opening. This conformational change can be measured as gating currents and is thought to be transferred to the pore domain via an annealing of the covalent link between voltage sensor and pore (S4-S5 linker) and the C terminus of the pore domain (S6). Upon prolonged depolarizations, the voltage de- pendence of the charge movement shifts to more hyperpolarized potentials. This mode shift had been linked to C-type inactivation but has recently been suggested to be caused by a relaxation of the voltage sensor itself. In this study, we identified two ShakerIR mutations in the S4-S5 linker (I384N) and S6 (F484G) that, when mutated, com- pletely uncouple voltage sensor movement from pore opening. Using these mutants, we show that the pore trans- fers energy onto the voltage sensor and that uncoupling the pore from the voltage sensor leads the voltage sensors to be activated at more negative potentials. This uncoupling also eliminates the mode shift occurring during pro- longed depolarizations, indicating that the pore influences entry into the mode shift. Using voltage-clamp fluo- rometry, we identified that the slow conformational change of the S4 previously correlated with the mode shift disappears when uncoupling the pore.
    [Show full text]
  • State-Dependent Inactivation of K + Currents in Rat Type II Alveolar Epithelial Cells
    State-dependent Inactivation of K + Currents in Rat Type II Alveolar Epithelial Cells THOMAS E. DECOURSEY From the Department of Physiology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612-3864 ABSTRACT Inactivation of K § channels responsible for delayed rectification in rat type II alveolar epithelial cells was studied in Ringer, 160 mM K-Ringer, and 20 mM Ca-Ringer. Inactivation is slower and less complete when the extracellular K + concentration is increased from 4.5 to 160 mM. Inactivation is faster and more complete when the extracellular Ca ~+ concentration is increased from 2 to 20 mM. Several observations suggest that inactivation is state-dependent. In each of these solutions depolarization to potentials near threshold results in slow and partial inactivation, whereas depolarization to potentials at which the K + conductance, gK, is fully activated results in maximal inactivation, suggesting that open channels inactivate more readily than closed channels. The time constant of current inacti- vation during depolarizing pulses is clearly voltage-dependent only at potentials where activation is incomplete, a result consistent with coupling of inactivation to activation. Additional evidence for state-dependent inactivation includes cumula- tive inactivation and nonmonotonic recovery from inactivation. A model like that proposed by C.M. Armstrong (1969.J. Gen. Physiol. 54: 553-575) for K + channel block by internal quaternary ammonium ions accounts for most of these proper- ties. The fundamental assumptions are: (a) inactivation is strictly coupled to activa- tion (channels must open before inactivating, and recovery from inactivation requires passage through the open state); (b) the rate of inactivation is voltage- independent.
    [Show full text]
  • The Episodic Ataxia Type 1 Mutation I262T Alters Voltage-Dependent
    www.nature.com/scientificreports OPEN The episodic ataxia type 1 mutation I262T alters voltage-dependent gating and disrupts protein Received: 05 October 2015 Accepted: 07 December 2015 biosynthesis of human Kv1.1 Published: 18 January 2016 potassium channels Szu-Han Chen1,*, Ssu-Ju Fu1,*, Jing-Jia Huang1 & Chih-Yung Tang1,2 Voltage-gated potassium (Kv) channels are essential for setting neuronal membrane excitability. Mutations in human Kv1.1 channels are linked to episodic ataxia type 1 (EA1). The EA1-associated mutation I262T was identified from a patient with atypical phenotypes. Although a previous report has characterized its suppression effect, several key questions regarding the impact of the I262T mutation on Kv1.1 as well as other members of the Kv1 subfamily remain unanswered. Herein we show that the dominant-negative effect of I262T on Kv1.1 current expression is not reversed by co-expression with Kvβ1.1 or Kvβ2 subunits. Biochemical examinations indicate that I262T displays enhanced protein degradation and impedes membrane trafficking of Kv1.1 wild-type subunits. I262T appears to be the first EA1 mutation directly associated with impaired protein stability. Further functional analyses demonstrate that I262T changes the voltage-dependent activation and Kvβ1.1-mediated inactivation, uncouples inactivation from activation gating, and decelerates the kinetics of cumulative inactivation of Kv1.1 channels. I262T also exerts similar dominant effects on the gating of Kv1.2 and Kv1.4 channels. Together our data suggest that I262T confers altered channel gating and reduced functional expression of Kv1 channels, which may account for some of the phenotypes of the EA1 patient.
    [Show full text]
  • Voltage-Dependent Gating of Single Sodium Channels from Mammalian Neuroblastoma Cells
    The Journal of Neuroscience, February 1987, 7(2): 418431 Voltage-Dependent Gating of Single Sodium Channels from Mammalian Neuroblastoma Cells Richard W. Aldrich” and Charles F. Stevens Section of Molecular Neurobiology, Yale University School of Medicine, New Haven, Connecticut 06510 Single sodium channel currents have been studied in cell- depends quite strongly on voltage. After a step increase in mem- attached patches from the mouse neuroblastoma cell line brane potential, the time course of current flow is, under many NlEl15. Distributions of open duration, latency until first circumstances, dominated by channel opening rather than some opening, and the average probability of a channel being other process (such as inactivation or simple channel closing). open after a voltage step, p(f), were analyzed and com- The results reported here, including an analysis of channel pared to predicted distributions from various kinetic models behavior during tail currents, confirm and extend our earlier for voltage-dependent gating. It was found that, over most observations (Aldrich and Stevens, 1983; Aldrich et al., 1983). of the voltage range under which channel gating occurs, the Perhaps our most striking finding is that activation processes, slow steps in gating are opening transitions and that in- rather than inactivation, dominate macroscopic sodium channel activation of open channels is significantly faster than the kinetics over a surprisingly large voltage range. What has tra- decline in p(f) (7J. This view of gating is confirmed by com- ditionally been recognized as the kinetics of inactivation, then, parison of the kinetics of ensemble averages of single-chan- appears to be a manifestation of a slow component of the ac- nel currents obtained from step- and tail-current records at tivation process in the type of sodium channels we have studied.
    [Show full text]