Direct inhibition of P/Q-type voltage-gated Ca2+ channels by Gem does not require a direct Gem/Cavβ interaction

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca2+ 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 Ca2+ channel beta subunits (Ca-v beta), 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 Ca2+ 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 beta but not the Gem/Ca-v beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca-v alpha(1)) in a Ca-v beta-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 Cava1 critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca-v alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca-v beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca-v alpha 1 that binds Ca-v beta). Our results challenge the prevailing view regarding the role of Ca-v beta in RGK inhibition of high voltage-activated Ca2+ channels and prompt a paradigm in which Gem directly binds and inhibits Ca-v beta-primed Ca-v alpha(1) on the plasma membrane.