Molecular determinants of the functional interaction between syntaxin and N-type Ca ²⁺ channel gating

Abstract

Syntaxin is a key presynaptic protein that binds to N-and P͞Q-type Ca 2؉ channels in biochemical studies and affects gating of these Ca 2؉ channels in expression systems and in synaptosomes. The present study was aimed at understanding the molecular basis of syntaxin modulation of N-type channel gating. Mutagenesis of either syntaxin 1A or the pore-forming ␣1B subunit of N-type Ca 2؉ channels was combined with functional assays of N-type channel gating in a Xenopus oocyte coexpression system and in biochemical binding experiments in vitro. Our analysis showed that the transmembrane region of syntaxin and a short region within the H3 helical cytoplasmic domain of syntaxin, containing residues Ala-240 and Val-244, appeared critical for the channel modulation but not for biochemical association with the ''synprint site'' in the II͞III loop of ␣1B. These results suggest that syntaxin and the ␣1B subunit engage in two kinds of interactions: an anchoring interaction via the II͞III loop synprint site and a modulatory interaction via another site located elsewhere in the channel sequence. The segment of syntaxin H3 found to be involved in the modulatory interaction would lie hidden within the four-helix structure of the SNARE complex, supporting the hypothesis that syntaxin's ability to regulate N-type Ca 2؉ channels would be enabled after SNARE complex disassembly after synaptic vesicle exocytosis.

Syntaxin 1A Modulates the Voltage-gated L-type Calcium Channel (Cav1.2) in a Cooperative Manner

“…The sequence specificity of the TMD is critical and greatly influences current amplitude. In contrast, TMD sequence alterations can be tolerated without alteration of Ca v 1.2 activation and inactivation, consistent with C-terminal mutations that do not alter the Sx1A effect on N-type channel inactivation (41).…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…22 Coexpression of syntaxin 1A (or 1B) with N-type or P/Q-type channels results in inhibition of channel activity, by virtue of a strong hyperpolarizing shift in the voltage dependence of steady state inactivation of the channel. 21,[67][68][69] A similar effect is observed upon co expression of N-type channels with SNAP-25. 67 For N-type channels, the inhibitory effects of syntaxin and SNAP-25 are reversed when both proteins are co expressed with the channel.…”

OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls Abscisic Acid–Mediated and Non-Abscisic Acid–Mediated Responses to Abiotic Stress

“…The H3 domain also is required for the syntaxin-mediated control of Ca 2ϩ channels in eukaryotic systems (Bezprozvanny et al, 2000). It is tempting to speculate that the conserved H3 domain of OSM1/SYP61 may interact with guard cell Ca 2ϩ channels to regulate ion fluxes and subsequent water movement that drives guard cell turgor changes and stomatal pore size .…”