Molecular determinants of the functional interaction between syntaxin and N-type Ca2+ channel gating

Syntaxin is a key presynaptic protein that binds to N- and P/Q-type Ca2+ ch annels in biochemical studies and affects gating of these Ca2+ channels in expression systems and in synaptosomes. The present study was aimed at unde rstanding the molecular basis of syntaxin modulation of N-type channel gati ng. Mutagenesis of either syntaxin 1A or the pore-forming cure subunit of N -type Ca2+ channels was combined with functional assays of N-type channel g ating in a Xenopus oocyte coexpression system and in biochemical binding ex periments in vitro. Our analysis showed that the transmembrane region of sy ntaxin and a short region within the H3 helical cytoplasmic domain of synta xin, containing residues Ala-240 and Val-244, appeared critical for the cha nnel modulation but not for biochemical association with the "synprint site " in the II/III loop of alpha (1B). These results suggest that syntaxin and the ale subunit engage in two kinds of interactions: an anchoring interact ion via the II/III loop synprint site and a modulatory interaction via anot her site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden wit hin the four-helix structure of the SNARE complex. supporting the hypothesi s that syntaxin's ability to regulate N-type Ca2+ channels would be enabled after SNARE complex disassembly after synaptic Vesicle exocytosis.