DOCSLIB.ORG

Identification of Cav2–Pkcβ and Cav2–NOS1 Complexes As Entities for Ultrafast Electrochemical Coupling

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of Cav2–Pkcβ and Cav2–NOS1 Complexes As Entities for Ultrafast Electrochemical Coupling Identification of Cav2–PKCβ and Cav2–NOS1 complexes as entities for ultrafast electrochemical coupling Cristina E. Constantina, Catrin S. Müllera, Michael G. Leitnerb, Wolfgang Bildla, Uwe Schultea,c, Dominik Oliverb, and Bernd Faklera,d,1 aInstitute of Physiology, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany; bDepartment of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps University, 35037 Marburg, Germany; cLogopharm GmbH, 79232 March-Buchheim, Germany; and dCenter for Biological Signaling Studies (BIOSS), 79104 Freiburg, Germany Edited by Bertil Hille, University of Washington School of Medicine, Seattle, WA, and approved April 21, 2017 (received for review October 2, 2016) Voltage-activated calcium (Cav) channels couple intracellular signal- properties of which distinctly shape amplitude and time course of 2+ + ing pathways to membrane potential by providing Ca ions as sec- the BKCa-mediated K currents (10, 11). ond messengers at sufficiently high concentrations to modulate In the presynapses of central neurons, the distance constraints + effector proteins located in the intimate vicinity of those channels. of local Ca2 signaling lead to embedding of the Cav channels Here we show that protein kinase Cβ (PKCβ) and brain nitric oxide into protein nano-environments containing a wide range of + synthase (NOS1), both identified by proteomic analysis as constituents Ca2 -controlled effector systems (6, 12, 13). In particular, pro- of the protein nano-environment of Cav2 channels in the brain, directly teomic analysis identified BKCa channels, the priming and fusion coassemble with Cav2.2 channels upon heterologous coexpression. machinery of neurotransmitter vesicles, as well as the enzymes Within Cav2.2–PKCβ and Cav2.2–NOS1 complexes voltage-triggered protein kinase C (PKCβ) and brain nitric oxide synthase 1 (NOS1) Ca2+ influx through the Cav channels reliably initiates enzymatic as constituents of the nano-environments of Cav2-type channels activity within milliseconds. Using BKCa channels as target sensors (Fig. 1B) (6). Although both PKCβ and NOS1 are known to be + for nitric oxide and protein phosphorylation together with high con- involved in Ca2 -operated signaling, their spatial relation to and + + centrations of Ca2 buffers showed that the complex-mediated Ca2 their control by Cav channels have not been elucidated. signaling occurs in local signaling domains at the plasma membrane. Here, we applied proteomic analyses together with FRET mea- Our results establish Cav2–enzyme complexes as molecular entities surements and electrophysiological recordings to investigate the struc- for fast electrochemical coupling that reliably convert brief mem- tural and functional characteristics of heterologously reconstituted brane depolarization into precisely timed intracellular signaling assemblies from Cav2.2 channels and either PKCβ (Cav2.2–PKCβ)or events in the mammalian brain. NOS1 (Cav2.2–NOS1). Our work establishes Cav2.2–PKCβ and Cav2.2–NOS1 as molecular entities for robust enzymatic activity that is + calcium channel | protein complex | Ca2 -dependent enzyme | NOS1 | PKC tightly controlled by the membrane potential, thus realizing fast and highly efficient electrochemical coupling. n CNS neurons, as in many other types of cells, a variety of Results intracellular signaling processes are tightly coupled to the I β 2+ membrane potential through voltage-gated calcium (Cav) chan- Complex Formation of PKC and NOS1 with Cav2.2 Channels. Ca - β nels. These channels, activated by the membrane depolarization dependent enzymes PKC and NOS1 have been identified as + occurring during action potentials, deliver calcium ions (Ca2 )to constituents of the extended protein networks making up the nano- environments of Cav2 channels [Cav2.1 (P/Q-type), Cav2.2 (N-type), the cytoplasm that subsequently serve as second messengers to + Cav2.3, R-type] (Fig. 1B) in the rodent brain (6). Within these drive the activity of Ca2 -dependent effector systems (1, 2). In the mammalian brain, these effectors serve a wide range of physio- Significance logical functions including the release of transmitter-loaded vesi- cles, regulation of excitability, gene transcription, neuronal growth, β β or plasticity of synaptic signal transduction. Nitric oxide synthase (NOS1) and protein kinase C (PKC ), + A hallmark feature of the Cav-triggered Ca2 signaling is its through their enzymatic activities, initiate a variety of in- tracellular signaling processes in the brain. For activation, both tight spatiotemporal constraints: mobile buffers restrict the diffusion + + + enzymes require intracellular calcium ions (Ca2 ), which, for of Ca2 , thus generating a local domain where the intracellular Ca2 2+ NOS1, may be provided via NMDA-type glutamate receptors. concentration ([Ca ]i) drops from hundreds of micromolars un- 2+ ’ μ Activation by voltage-gated Ca (Cav) channels has remained derneath the Cav channel s inner vestibule to values below 10 Mat elusive. Here we report that both PKCβ and NOS1 form bi- a distance of several tens of nanometers (3–5). The spatial + + molecular complexes with Cav2.2 channels, thus placing 2 “ 2 + profile of these local [Ca ]i domains, often referred to as Ca themselves within less than 10 nm of a Ca2 source. Within ” nano-domains, is predominantly controlled by the binding the framework of the channel–enzyme complexes, PKCβ and 2+ properties of the Ca buffers, whereas the temporal character- NOS1 are reliably activated even by periods of Ca2+ influx as 2+ istics of these local [Ca ]i domains are largely determined by the short as 0.8 ms occurring during action potentials. Our results gating of the Cav channels (5). Bimolecular complexes of Cav establish Cav2–enzyme complexes as molecular entities for fast 2+ + channels and BK-type Ca -activated K channels (BKCa)are and precisely timed electrochemical coupling. + NEUROSCIENCE prototypic Ca2 nano-domains reflecting both spatial dimensions + and operation principles of local Ca2 signaling (Fig. 1A)(4,6,7–9). Author contributions: C.E.C. and B.F. designed research; C.E.C., C.S.M., M.G.L., W.B., and U.S. performed research; C.E.C., C.S.M., M.G.L., W.B., U.S., D.O., and B.F. analyzed data; and B.F. In this bimolecular arrangement, both channels are separated wrote the paper. ∼ 2+ ≥ μ by only 10 nm, thus guaranteeing a [Ca ]i of 10 M required The authors declare no conflict of interest. for robust activation of BKCa at physiological voltages. Moreover, 2+ This article is a PNAS Direct Submission. thesehighvaluesfor[Ca ]i are provided during individual action 1 2+ To whom correspondence should be addressed. Email: [email protected] potentials and even in the presence of high concentrations of Ca freiburg.de. – BIOPHYSICS AND buffers (8, 10). The output of the Cav BKCa complex(es) is strictly This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. controlled by the respective Cav channel subtype the gating 1073/pnas.1616394114/-/DCSupplemental. COMPUTATIONAL BIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1616394114 PNAS | May 30, 2017 | vol. 114 | no. 22 | 5707–5712 Downloaded by guest on September 30, 2021 A levels between brain and cultured cells, see Fig. S1B). Membrane BKCa fractions prepared from the respective cells were equilibrated extra with antibodies (ABs) against the three target proteins (anti- Cav2.2, anti-PKCβ, and anti-NOS1)(Materials and Methods), and the eluates of these APs were subjected to nano-LC MS/MS, intra which provided information on both the identity and the amount Cav 1 Cav of any protein therein (6, 14, 15). Quantitative evalution of these 10 nm APs showed that the anti-Cav2.2 AB purified the subunits of its 10 nm [Ca2+] profile target channel in all experiments and, in addition, effectively retained the coexpressed PKCβ (Fig. 1C, Left) and NOS1 (Fig. 1C, Right). Conversely, the ABs specifically targeting either of the two enzymes copurified all Cav channel subunits (Fig. 1C, B Proteome of Cav2 networks bars in black and gray). whole rat brain Ca2+-dependent effectors Together, these results indicate that both PKCβ and NOS1 are Classification/Function Protein ID Acc.Name (UniProtKB) able to coassemble with Cav2.2 channels without the addition of further linker protein(s), most likely by formation of bimolecular Ion channel BKCa KCMA1 – Enzyme(s) channel enzyme complexes. kinases PKC , CaMKII KPCA KCC2B PKC CaMKII KPCB KCC2D – KPCG KCC2G Operation of Cav2.2 NOS1 Complexes. Next we investigated the PKC CaMKII – phosphatase PP2B PP2BA functional properties of the Cav2.2 NOS1 assemblies. For this NO synthase NOS1 NOS1 purpose, we applied a genetically encoded reporter system for Vesicle fusion Syntaxin, SNAP25, VAMP2, STX1A, SNP25, VAMP2, nitric oxide (NO) that is based on NO-mediated synthesis of Synaptotagmin2/7 SYT2/7 cGMP and subsequent cGMP-triggered FRET between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) C Cav2.2 + Cav 1 + 2 in an indicator protein (16). When coexpressed with NOS1 and APs from heterologous +/- PKC or NOS1 A Upper co-expressions Cav2.2 Cav2.2 in the aforementioned CHO cell line (Fig. 2 , ), CHO cells – Cav 1 this indicator enabled monitoring of NO generated by Cav2.2 2 NOS1 complexes in response to voltage pulses by means of 10000 PKC combined patch-clamp and fluorescence measurements. NOS1 Fig. 2A, Lower,andB illustrate the results of such measure- 1000 ments obtained during a single-voltage-step experiment in a CHO + cell that was dialyzed with high concentrations of the Ca2 buffer 100 + EGTA (10 mM; applied through the patch pipette): The Ca2 ions 10 conducted inward through activated Cav2.2 channels during a hyperpolarization step to −80 mV (Cav2.2 currents, Fig. 2A, 1 Lower) triggered a transient increase in YFP/CFP emission, reflecting NO production (Fig. 2B). This increase in FRET signal, Abundance ratio (+Enz / -Enz) 0.1 which was independent of the Cav2 channel density in the plasma -AP -AP membrane (Fig. S2), was observed only with NOS1 in wild-type anti-Cav2 anti-PKC-AP anti-Cav2 anti-NOS1-AP form, but not with a NOS1 mutant deprived of enzymatic activity Fig.
Load more

Recommended publications
  • Targeted Genes and Methodology Details for Neuromuscular Genetic Panels
    Targeted Genes and Methodology Details for Neuromuscular Genetic Panels Reference transcripts based on build GRCh37 (hg19) interrogated by Neuromuscular Genetic Panels Next-generation sequencing (NGS) and/or Sanger sequencing is performed Motor Neuron Disease Panel to test for the presence of a mutation in these genes. Gene GenBank Accession Number Regions of homology, high GC-rich content, and repetitive sequences may ALS2 NM_020919 not provide accurate sequence. Therefore, all reported alterations detected ANG NM_001145 by NGS are confirmed by an independent reference method based on laboratory developed criteria. However, this does not rule out the possibility CHMP2B NM_014043 of a false-negative result in these regions. ERBB4 NM_005235 Sanger sequencing is used to confirm alterations detected by NGS when FIG4 NM_014845 appropriate.(Unpublished Mayo method) FUS NM_004960 HNRNPA1 NM_031157 OPTN NM_021980 PFN1 NM_005022 SETX NM_015046 SIGMAR1 NM_005866 SOD1 NM_000454 SQSTM1 NM_003900 TARDBP NM_007375 UBQLN2 NM_013444 VAPB NM_004738 VCP NM_007126 ©2018 Mayo Foundation for Medical Education and Research Page 1 of 14 MC4091-83rev1018 Muscular Dystrophy Panel Muscular Dystrophy Panel Gene GenBank Accession Number Gene GenBank Accession Number ACTA1 NM_001100 LMNA NM_170707 ANO5 NM_213599 LPIN1 NM_145693 B3GALNT2 NM_152490 MATR3 NM_199189 B4GAT1 NM_006876 MYH2 NM_017534 BAG3 NM_004281 MYH7 NM_000257 BIN1 NM_139343 MYOT NM_006790 BVES NM_007073 NEB NM_004543 CAPN3 NM_000070 PLEC NM_000445 CAV3 NM_033337 POMGNT1 NM_017739 CAVIN1 NM_012232 POMGNT2
    [Show full text]
  • S100P Interacts with P53 While Pentamidine Inhibits This Interaction
    biomolecules Article S100P Interacts with p53 while Pentamidine Inhibits This Interaction Revansiddha H. Katte 1 , Deepu Dowarha 1 , Ruey-Hwang Chou 2,3 and Chin Yu 1,* 1 Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan; [email protected] (R.H.K.); [email protected] (D.D.) 2 Graduate Institute of Biomedical Sciences and Center for Molecular Medicine, China Medical University, Taichung 40402, Taiwan; [email protected] 3 Department of Biotechnology, Asia University, Taichung 41354, Taiwan * Correspondence: [email protected]; Tel.: +886-963-780-784; Fax: +886-35-711082 Abstract: S100P, a small calcium-binding protein, associates with the p53 protein with micromolar affinity. It has been hypothesized that the oncogenic function of S100P may involve binding-induced inactivation of p53. We used 1H-15N HSQC experiments and molecular modeling to study the molecular interactions between S100P and p53 in the presence and absence of pentamidine. Our experimental analysis indicates that the S100P-53 complex formation is successfully disrupted by pentamidine, since S100P shares the same binding site for p53 and pentamidine. In addition, we showed that pentamidine treatment of ZR-75-1 breast cancer cells resulted in reduced proliferation and increased p53 and p21 protein levels, indicating that pentamidine is an effective antagonist that interferes with the S100P-p53 interaction, leading to re-activation of the p53-21 pathway and inhibition of cancer cell proliferation. Collectively, our findings suggest that blocking the association between S100P and p53 by pentamidine will prevent cancer progression and, therefore, provide a new avenue for cancer therapy by targeting the S100P-p53 interaction.
    [Show full text]
  • Mapping Protein Interactions of Sodium Channel Nav1.7 Using 2 Epitope-Tagged Gene Targeted Mice
    bioRxiv preprint doi: https://doi.org/10.1101/118497; this version posted March 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Mapping protein interactions of sodium channel NaV1.7 using 2 epitope-tagged gene targeted mice 3 Alexandros H. Kanellopoulos1†, Jennifer Koenig1, Honglei Huang2, Martina Pyrski3, 4 Queensta Millet1, Stephane Lolignier1, Toru Morohashi1, Samuel J. Gossage1, Maude 5 Jay1, John Linley1, Georgios Baskozos4, Benedikt Kessler2, James J. Cox1, Frank 6 Zufall3, John N. Wood1* and Jing Zhao1†** 7 1Molecular Nociception Group, WIBR, University College London, Gower Street, 8 London WC1E 6BT, UK. 9 2Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, The 10 Old Road Campus, Oxford, OX3 7FZ, UK. 11 3Center for Integrative Physiology and Molecular Medicine, Saarland University, 12 Kirrbergerstrasse, Bldg. 48, 66421 Homburg, Germany. 13 4Division of Bioscience, University College London, Gower Street, London WC1E 6BT, UK.14 15 †These authors contributed equally to directing this work. 16 *Correspondence author. Tel: +44 207 6796 954; E-mail: [email protected] 17 **Corresponding author: Tel: +44 207 6790 959; E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/118497; this version posted March 20, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 Abstract 19 The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. 20 Besides action potential propagation, NaV1.7 regulates neurotransmitter release, 21 integrates depolarizing inputs over long periods and regulates transcription.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Familial Hyperkalemic Hypertension
    Disease of the Month Familial Hyperkalemic Hypertension Juliette Hadchouel,* Ce´line Delaloy,* Se´bastien Faure´,† Jean-Michel Achard,† and Xavier Jeunemaitre* *Colle`ge de France, Paris; INSERM U36, Paris; AP-HP, Department of Genetics, Hoˆpital Europe´en Georges Pompidou; University Paris-Descartes, Faculty of Medicine, Paris, France; and †Division of Nephrology and Department of Physiology, Limoges University Hospital, Limoges, France J Am Soc Nephrol 17: 208–217, 2006. doi: 10.1681/ASN.2005030314 amilial hyperkalemic hypertension (FHHt) syndrome biochemical abnormalities and age or BP, which seemed to (1,2), also known as Gordon syndrome (3) or pseudohy- depend primarily on age in affected individuals (11). This F poaldosteronism type 2 (4), is a rare inherited form of phenotypic variability, associated with sensitivity to thia- low-renin hypertension associated with hyperkalemia and hy- zides—which are widely used in hypertension—and with a low perchloremic metabolic acidosis in patients with a normal GFR probability of this rare disease’s being recognized by most (OMIM no. 145260). This monogenic form of arterial hyperten- doctors may have led to an underestimation of its frequency. sion has excited new interest since the discovery of a new, The mode of inheritance of the disease is consistent with unsuspected molecular pathway that is responsible for both the autosomal dominant transmission in most, if not all, of the biochemical abnormalities and the increase in BP observed. pedigrees reported. However, we have identified two families Genetic analysis has led to the identification of mutations in in which the parents of the affected individuals were first two genes that belong to a new family of kinases, the WNK cousins, suggesting possible autosomal recessive transmission.
    [Show full text]
  • Mapping Protein Interactions of Sodium Channel
    Published online: January 15, 2018 Resource Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene-targeted mice Alexandros H Kanellopoulos1,†, Jennifer Koenig1,†, Honglei Huang2, Martina Pyrski3 , Queensta Millet1, Stéphane Lolignier1,4, Toru Morohashi1, Samuel J Gossage1, Maude Jay1, John E Linley1,5, Georgios Baskozos6 , Benedikt M Kessler2, James J Cox1, Annette C Dolphin7, Frank Zufall3, John N Wood1,* & Jing Zhao1,** Abstract Introduction The voltage-gated sodium channel NaV1.7 plays a critical role in Pain is a major clinical problem. A 2012 National Health Interview pain pathways. We generated an epitope-tagged NaV1.7 mouse Survey (NHIS) in the United States revealed that 25.3 million adults that showed normal pain behaviours to identify channel-inter- (11.2%) suffered from daily (chronic) pain and 23.4 million acting proteins. Analysis of NaV1.7 complexes affinity-purified (10.3%) reported severe pain within a previous 3-month period under native conditions by mass spectrometry revealed 267 (Nahin, 2015). Many types of chronic pain are difficult to treat as proteins associated with Nav1.7 in vivo. The sodium channel b3 most available drugs have limited efficacy and can cause side (Scn3b), rather than the b1 subunit, complexes with Nav1.7, and effects. There is thus a huge unmet need for novel analgesics we demonstrate an interaction between collapsing-response medi- (Woodcock, 2009). Recent human and animal genetic studies have ator protein (Crmp2) and Nav1.7, through which the analgesic drug indicated that the voltage-gated sodium channel (VGSC) NaV1.7 lacosamide regulates Nav1.7 current density. Novel NaV1.7 protein plays a crucial role in pain signalling (Nassar et al, 2004; Cox et al, interactors including membrane-trafficking protein synaptotag- 2006; Dib-Hajj et al, 2013), highlighting NaV1.7 as a promising drug min-2 (Syt2), L-type amino acid transporter 1 (Lat1) and trans- target for development of novel analgesics (Habib et al, 2015; membrane P24-trafficking protein 10 (Tmed10) together with Emery et al, 2016).
    [Show full text]
  • Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease
    Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease Sarah Bergen Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Medical Genetics Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1940 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. Genes and Symptoms of Schizophrenia: Modifiers, Networks, and Interactions in Complex Disease A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University by: Sarah E. Bergen B.A., Macalester College, 2000 M.S., University of Pittsburgh, 2004 Director: Kenneth S. Kendler, M.D. Distinguished Professor, Departments of Psychiatry and Human and Molecular Genetics Virginia Commonwealth University Richmond, VA September, 2009 Acknowledgements First, I want to thank my parents whose unconditional love and only slightly conditional support has carried me through every bump and rough patch on my journey through grad school (and life). None of my work would have been accomplished without all you have done for me, and I am profoundly grateful. I have been extremely fortunate in my experiences here to not only have been quite productive scientifically, but also to have enjoyed the graduate school experience. This is almost entirely due to the brilliant and kind people I have had the opportunity to work with.
    [Show full text]
  • Calcium Control of Neurotransmitter Release
    Downloaded from http://cshperspectives.cshlp.org/ on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Calcium Control of Neurotransmitter Release Thomas C. Su¨dhof Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305 Correspondence: [email protected] Upon entering a presynaptic terminal, an action potential opens Ca2þ channels, and transiently increases the local Ca2þ concentration at the presynaptic active zone. Ca2þ then triggers neurotransmitter release within a few hundred microseconds by activating synaptotagmins Ca2þ. Synaptotagmins bind Ca2þ via two C2-domains, and transduce the Ca2þ signal into a nanomechanical activation of the membrane fusion machinery; this acti- vation is mediated by the Ca2þ-dependent interaction of the synaptotagmin C2-domains with phospholipids and SNARE proteins. In triggering exocytosis, synaptotagmins do not act alone, but require an obligatory cofactor called complexin, a small protein that binds to SNARE complexes and simultaneously activates and clamps the SNARE complexes, thereby positioning the SNARE complexes for subsequent synaptotagmin action. The con- served function of synaptotagmins and complexins operates generally in most, if not all, Ca2þ-regulated forms of exocytosis throughout the body in addition to synaptic vesicle exo- cytosis, including in the degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release. ynaptic transmission is initiated when an in the brainstem (Fig. 1B; reviewed in Mein- Saction potential invades a nerve terminal, renken et al. 2003). Overall, these high-resolution opening Ca2þ channels, which gate a highly electrophysiological studies on neurotrans- localized, transient increase in intracellular mitter release revealed that a presynaptic action Ca2þ at the active zone (Fig.
    [Show full text]
  • Gangliosides Interact with Synaptotagmin to Form the High-Affinity Receptor Complex for Botulinum Neurotoxin B
    Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B Alessandra Floresa,b,1, Jorge Ramirez-Francoa,b,1, Richard Desplantesa,b, Kévin Debreuxa,b, Géraldine Ferraccib,c, Florian Wernerta,b, Marie-Pierre Blanchardb,c, Yves Mauleta,b, Fahamoe Youssoufa,b, Marion Sangiardia,b, Cécile Iborraa,b, Michel Robert Popoffd, Michael Seagara,b, Jacques Fantinia,b, Christian Lévêquea,b, and Oussama El Fara,b,2 aUnité de Neurobiologie des Canaux Ioniques et de la Synapse, INSERM UMR_S 1072, 13015 Marseille, France; bAix-Marseille Université, 13015 Marseille, France; cInstitut de Neurophysiopathologie, CNRS UMR 7051, 13015 Marseille, France; and dBacterial Toxins, Équipe de Recherche Labellisée 6002, Institut Pasteur, 75015 Paris, France Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 1, 2019 (received for review May 15, 2019) Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals synaptic vesicle proteins that become accessible at the presynaptic by binding to 2 receptor components: a polysialoganglioside, membrane upon exocytosis. BoNT/A and E bind to the synaptic predominantly GT1b, and synaptotagmin 1/2. It is widely thought vesicle protein 2 (SV2), while BoNT/B binds synaptotagmin (SYT that BoNT/B initially binds to GT1b then diffuses in the plane of isoforms 1 and 2). the membrane to interact with synaptotagmin. We have addressed Gangliosides are amphiphilic glycosphingolipid molecules the hypothesis that a GT1b–synaptotagmin cis complex forms the predominantly anchored, via their ceramide group, in the outer BoNT/B receptor. We identified a consensus glycosphingolipid-binding leaflet of plasma membranes. Their extracellular polar part is motif in the extracellular juxtamembrane domain of synaptotagmins composed of several characteristic sugar molecules with the 1/2 and confirmed by Langmuir monolayer, surface plasmon reso- negatively charged sialic acids at precise positions.
    [Show full text]
  • Role of Endothelium, Acetylocholine and Calcium Ions in Bay K8644- and Kcl-Induced Contraction
    914 MOLECULAR MEDICINE REPORTS 8: 914-918, 2013 Role of endothelium, acetylocholine and calcium ions in Bay K8644- and KCl-induced contraction KATARZYNA SZADUJKIS-SZADURSKA*, GRZEGORZ GRZESK*, LESZEK SZADUJKIS-SZADURSKI, MARTA GAJDUS, BARTOSZ MALINOWSKI and MICHAL WICINSKI Department of Pharmacology and Therapeutics, Collegium Medicum Nicolaus Copernicus University, Bydgoszcz 85-094, Poland Received February 17, 2013; Accepted June 13, 2013 DOI: 10.3892/mmr.2013.1574 Abstract. The aim of this study was to establish the involve- the factors examined, resulting in an influx of Ca2+ into the ment of acetylcholine (Ach) and calcium ions in modulating cell. contractions induced by Bay K8644 (an agonist of calcium channels located in the cell membrane) and KCl (at depola- Introduction rizing concentrations), and also to examine the importance of the vascular endothelium in the activity of Bay K8644. The The structure of the arteries is important as serve as a target study was performed on perfused Wistar rat tail arteries. for a number of substances that regulate smooth muscle Contraction induced by Bay K8644 with the participation of tension. The endothelium is a cell layer that lines the inside intracellular (in calcium-free physiological salt solution, FPSS) of blood vessels, and also produces and releases mediators and extracellular (in physiological salt solution, PSS, following that modulate the contraction of arteries (1,2). the emptying of the cellular Ca2+ stores) pools of Ca2+ and the Endothelial damage occurs in the course of various path- addition of nitro-L-arginine (L-NNA; nitric oxide synthase ological processes, particularly atherosclerosis, and leads inhibitor) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one to vascular disorders with regard to diameter regulation, (ODQ; an inhibitor of soluble guanylyl cyclase) was studied.
    [Show full text]
  • The Molecular Machinery of Neurotransmitter Release Nobel Lecture, 7 December 2013
    The Molecular Machinery of Neurotransmitter Release Nobel Lecture, 7 December 2013 by Thomas C. Südhof Dept. of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University, USA. 1. THE NEUROTRANSMITTER RELEASE ENIGMA Synapses have a long history in science. Synapses were frst functionally demon- strated by Emil duBois-Reymond (1818–1896), were morphologically identifed by classical neuroanatomists such as Rudolf von Kölliker (1817–1905) and San- tiago Ramon y Cajal (1852–1934), and named in 1897 by Michael Foster (1836– 1907). Although the chemical nature of synaptic transmission was already sug- gested by duBois-Reymond, it was long disputed because of its incredible speed. Over time, however, overwhelming evidence established that most synapses use chemical messengers called neurotransmitters, most notably with the pioneer- ing contributions by Otto Loewi (1873–1961), Henry Dale (1875–1968), Ulf von Euler (1905–1983), and Julius Axelrod (1912–2004). In parallel, arguably the most important advance to understanding how synapses work was provided by Bernard Katz (1911–2003), who elucidated the principal mechanism of syn- aptic transmission (Katz, 1969). Most initial studies on synapses were carried out on the neuromuscular junction, and central synapses have only come to the fore in recent decades. Here, major contributions by many scientists, including George Palade, Rodolfo Llinas, Chuck Stevens, Bert Sakmann, Eric Kandel, and Victor Whittaker, to name just a few, not only confrmed the principal results obtained in the neuromuscular junction by Katz, but also revealed that synapses 259 6490_Book.indb 259 11/4/14 2:29 PM 260 The Nobel Prizes exhibit an enormous diversity of properties as well as an unexpected capacity for plasticity.
    [Show full text]
  • Sildenafil Reverses O2 Constriction of the Rabbit Ductus Arteriosus by Inhibiting Type 5 Phosphodiesterase and Activating Bkca C
    0031-3998/02/5201-0019 PEDIATRIC RESEARCH Vol. 52, No. 1, 2002 Copyright © 2002 International Pediatric Research Foundation, Inc. Printed in U.S.A. Sildenafil Reverses O2 Constriction of the Rabbit Ductus Arteriosus by Inhibiting Type 5 Phosphodiesterase and Activating BKCa Channels BERNARD THÉBAUD, EVANGELOS MICHELAKIS, XI-CHEN WU, GWYNETH HARRY, KYOKO HASHIMOTO, AND STEPHEN L. ARCHER Department of Medicine (Cardiology) and the Vascular Biology Group [B.T., E.M., X.-C.W., G.H., K.H., S.L.A.], Department of Physiology [S.L.A.], University of Alberta, Edmonton, Alberta, T6G 2S2, Canada ABSTRACT Oxygen constriction causes functional closure of the ductus the DA by increasing soluble guanylyl-cyclase–derived cGMP arteriosus (DA) at birth. Although DA closure is crucial for levels and thereby activating calcium-sensitive potassium chan- postnatal adaptation, patency of the DA is critical for survival of nels, causing membrane hyperpolarization. Sildenafil, already newborns with duct-dependent cardiac malformations. In these approved for human usage, might be an alternative or a useful cases, DA patency is achieved by i.v. infusion of prostaglandin adjunct to prostaglandin E1 as a bridge to cardiac surgery. E1, which, though effective, is often associated with complica- (Pediatr Res 52: 19–24, 2002) tions. We hypothesized that sildenafil, a specific phosphodiester- ase type 5 inhibitor, is an effective DA vasodilator. In isolated Abbreviations DA rings from term (d 30) fetal rabbits, sildenafil (10Ϫ6–10Ϫ4 4-AP, 4-aminopyridine Ϫ7 Ϫ5 M) and diethylamine NONOate (10 –10 M) induced dose- BKCa, large conductance calcium-sensitive potassium channels dependent relaxation of oxygen-constricted DA (Ϫ52 Ϯ 4% and cGMP, 3',5'-cyclic GMP Ϫ51 Ϯ 6%, respectively) that was inhibited by the soluble DA, ductus arteriosus guanylyl-cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxa- DASMC, DA smooth muscle cells lin-1-one (5 ϫ 10Ϫ5 M).
    [Show full text]