Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner.
Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.
Institute of Life Sciences, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Hadar
H
Arien
Ofer
O
Wiser
Isaiah T
IT
Arkin
Hadas
H
Leonov
Daphne
D
Atlas
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D001152:Arsenicals / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000187:drug effects*; D002462:Cell Membrane / Q000737:chemistry; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004112:Dimercaprol / Q000494:pharmacology; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004553:Electric Conductivity; D005260:Female; D015870:Gene Expression; D008564:Membrane Potentials; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry; D050827:Syntaxin 1; D014162:Transfection; D014982:Xenopus laevis
D000954:Antigens, Surface; D001152:Arsenicals; D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D004220:Disulfides; C419385:L-type calcium channel alpha(1C); D009419:Nerve Tissue Proteins; C494213:Stx1a protein, rat; D013438:Sulfhydryl Compounds; D050827:Syntaxin 1; D004112:Dimercaprol; C029341:oxophenylarsine; D003545:Cysteine; D002118:Calcium