Direct inhibition of P/Q-type voltage-gated [Ca.sup.2+] channels by Gem does not require a direct Gem/[Ca.sub.v][beta] interaction

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) [Ca.sup.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.sup.2+] channel [beta] subunits ([Ca.sub.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 [Ca.sup.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.sub.v][beta] but not the Gem/[Ca.sub.v][beta] interaction, and that Gem coimmunoprecipitates with the P/Q channel [[alpha].sub.1] subunit ([Ca.sub.v][[alpha].sub.1]) in a [Ca.sub.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 [Ca.sub.v][[alpha].sub.1] critical for Gem inhibition. Exchanging this region between P/Q and T channel [Ca.sub.v][[alpha].sub.1] abolishes Gem inhibition of P/Q channels and confers [Ca.sub.v][beta]-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of [Ca.sub.v][[alpha].sub.1] that binds [Ca.sub.v][beta]). Our results challenge the prevailing view regarding the role of [Ca.sub.v][beta] in RGK inhibition of high voltage-activated [Ca.sup.2+] channels and prompt a paradigm in which Gem directly binds and inhibits [Ca.sub.v][beta]-primed [Ca.sub.v][[alpha].sub.1] on the plasma membrane. [beta] subunit | electrophysiology | modulation | Rem, Rem2, Rad, and Gem/Kir proteins | T-type [Ca.sup.2+] channels doi/ 10.1073/pnas.1007543107