Syntaxin 1A (Sx1A) modifies the activity of voltagegated Ca 2؉ 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 Ca 2؉ channel, Ca v 1.2, 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 ␣ 1 1.2/␣ 2 ␦1/ ␤2a 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 Ca 2؉ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca 2؉ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca 2؉ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.Signal transduction in the synapse is initiated by neurotransmitter release as a consequence of fusion that takes place between synaptic vesicles and the plasma membrane. It is commonly accepted that at the heart of this tightly regulated, multifarious process lies the ternary complex formed by the synaptic proteins syntaxin 1A (Sx1A), 1 SNAP-25, and synaptobrevin II (1-3). This ternary complex is thought to be responsible for juxtaposing the synaptic vesicle and the plasma membrane prior to membrane fusion, which involves the binding of soluble N-ethylmaleimide-sensitive factor (NSF) (1-3). It is for this reason that the above-mentioned ternary complex is referred to as the SNARE (receptor for soluble NSF attachment proteins) complex.Analysis of ternary complexes formed by the full-length proteins revealed that the C-terminal transmembrane domains (TMDs) of both Sx1A and synaptobrevin II were protected from trypsin digestion (4). Moreover, inclusion of these TMDs increased the stability of the ternary complexes and affected the ability of the cytoplasmic domains to join other proteins (4, 5). Co-reconstituted into liposomes, synaptobrevin II and syntaxin 1A formed a stable binary complex that required the presence of th...