Molecular Determinants of Voltage-Gated Calcium Channel Inhibition by Gem

High voltage-activated (HVA) Ca2+ channels, including L-, N- and P/Q-type channels, are potently inhibited by the Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins. This inhibition was widely thought to depend on the direct association between RGK proteins and the β subunit (Cavβ) of HVA Ca2+ channels, but we recently found that P/Q channel inhibition by Gem protein in inside-out membrane patches required Cavβ but not the Gem-Cavβ interaction, and that Gem coimmunoprecipitated with the P/Q channel α1 subunit in a Cavβ-independent manner. Thus, we proposed that direct interactions between Gem and the α1 subunit (Cavα1) of HVA Ca2+ channels are critical for Gem inhibition. In this study, we investigate the molecular determinants on Cavα1 that are important for Gem inhibition. We construct chimeras between the α1 subunit of P/Q channels and the Gem-insensitive low voltage-activated T-type channels, which do not bind or require Cavβ. We find that grafting the α-interaction domain (AID), the high-affinity Cavβ binding site on Cavα1, or grafting the entire AID-containing I-II loop from P/Q channel into T-channels (TPQ I-II loop), is sufficient for Cavβ binding and gating regulation but does not bestow RGK inhibition. However, adding the first two transmembrane segments (IIS1-IIS2) of the second homology repeat onto the TPQ I-II loop construct, produced a Gem-sensitive T-channel. This inhibition persists with non-interacting Gem and Cavβ mutants, indicating that the Cavβ-Gem interaction is not necessary, just as in the case of Gem inhibition of P/Q channels. In complementary experiments, substituting only IIS1-IIS2 of P/Q channel α1 with that of T-channel's severely attenuates RGK inhibition. This supports a paradigm in which Gem directly binds and inhibits Cavβ-primed Cavα1 on the plasma membrane. We are currently investigating the role of other Cavα1 regions in Gem inhibition.