Direct inhibition of P/Q-type voltage-gated Ca 2+ channels by Gem does not require a direct Gem/Ca v β interaction

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca 2+ channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca 2+ channel β subunits (Ca v β), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca 2+ channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca v β but not the Gem/Ca v β interaction, and that Gem coimmunoprecipitates with the P/Q channel α 1 subunit (Ca v α 1 ) in a Ca v β-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca v α 1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca v α 1 abolishes Gem inhibition of P/Q channels and confers Ca v β-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca v α 1 that binds Ca v β). Our results challenge the prevailing view regarding the role of Ca v β in RGK inhibition of high voltage-activated Ca 2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca v β-primed Ca v α 1 on the plasma membrane.