Neuroscience 2001 Jan 01;1042:599-607. doi: 10.1016/s0306-4522(01)00083-5.

The transmembrane domain of syntaxin 1A negatively regulates voltage-sensitive Ca(2+) channels.

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Syntaxin 1A has a pronounced inhibitory effect on the activation kinetics and current amplitude of voltage-gated Ca(2+) channels. This study explores the molecular basis of syntaxin interaction with N- and Lc-type Ca(2+) channels by way of functional assays of channel gating in a Xenopus oocytes expression system. A chimera of syntaxin 1A and syntaxin 2 in which the transmembrane domain of syntaxin 2 replaced the transmembrane of syntaxin 1A (Sx1-2), significantly reduced the rate of activation of N- and Lc-channels. This shows a similar effect to that demonstrated by syntaxin 1A, though the current was not inhibited. The major sequence differences at the transmembrane of the syntaxin isoforms are that the two highly conserved cysteines Cys 271 and Cys 272 in syntaxin 1A correspond to the valines Val 272 and Val 273 in syntaxin 2 transmembrane. Mutating either cysteines in Sx1-1 (syntaxin 1A) to valines, did not affect modulation of the channel while a double mutant C271/272V was unable to regulate inward current. Transfer of these two cysteines to the transmembrane of syntaxin 2 by mutating Val 272 and Val 273 to Cys 272 and Cys 273 led to channel inhibition. When cleaved by botulinum toxin, the syntaxin 1A fragments, amino acids 1-253 and 254-288, which includes the transmembrane domain, were both unable to inhibit current amplitude but retained the ability to modify the activation kinetics of the channel. A full-length syntaxin 1A and the integrity of the two cysteines within the transmembrane are crucial for coordinating Ca(2+) entry through the N- and Lc-channels. These results suggest that upon membrane depolarization, the voltage-gated N- and Lc-type Ca(2+)-channels signal the exocytotic machinery by interacting with syntaxin 1A at the transmembrane and the cytosolic domains. Cleavage with botulinum toxin disrupts the coupling of the N- and Lc-type channels with syntaxin 1A and abolishes exocytosis, supporting the hypothesis that these channels actively participate in Ca(2+) regulated secretion.

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Species referenced: Xenopus laevis
Genes referenced: stx1a