G protein modulation of N-type calcium channels is facilitated by physicalinteractions between syntaxin 1A and G beta gamma

The direct modulation of N-type calcium channels by G protein beta gamma su bunits is considered a key factor in the regulation of neurotransmission. S ome of the molecular determinants that govern the binding interaction of N- type channels and G beta gamma have recently been identified (see, Le., Zam poni, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997 ) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presyn aptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by G beta gamma. When syntaxin 1A was c oexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inacti vation potential, as well as a pronounced tonic G protein inhibition as ass essed by its reversal by strong membrane depolarizations. This tonic inhibi tion was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the en hanced G; protein modulation. However, when G protein beta gamma subunits w ere concomitantly coexpressed, the toxin became ineffective in removing G p rotein inhibition, suggesting that syntaxin LA optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that G beta gamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with G beta gamma and the synprint motif of the N- type channel II-III linker. Taken together, our experiments suggest a mecha nism by which syntaxin IA mediates a colocalization of G protein beta gamma subunits and N-type calcium channels, thus resulting in more effective G p rotein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the s tructural specialization of the presynaptic terminal.