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G Protein Modulation of N-type Calcium Channels Is Facilitated by Physical Interactions between Syntaxin 1A and Gβγ

G Protein Modulation of N-type Calcium Channels Is Facilitated by Physical Interactions between Syntaxin 1A and Gβγ*

Abstract

The direct modulation of N-type calcium channels by G protein βγ subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gβγ have recently been identified (see, i.e., Zamponi, 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 presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gβγ. When syntaxin 1A was coexpressed with (N-type) α1B + α2-δ + β1b channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein βγ subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gβγ physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gβγ and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein βγ subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.

  • Abbreviations:
    BTC1
    botulinum toxin C1
    EGFP
    enhanced green fluorescent protein
    GST
    glutathioneS-transferase
    PMSF
    phenylmethylsulfonyl fluoride
    PAGE
    polyacrylamide gel electrophoresis
    DMEM
    Dulbecco's modified Eagle's medium
    PBS
    phosphate-buffered saline
    PBST
    phosphate-buffered saline plus Tween 20
    PCR
    polymerase chain reaction
    MOPS
    4-morpholinepropanesulfonic acid
    SNARE
    solubleN-ethylmaleimide-sensitive factor attachment protein receptor
    • Received October 13, 1999.
    • Revision received December 9, 1999.
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