Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels.

Genetically encoded inhibitors for voltage-dependent Ca (Ca) channels (GECCIs) are useful research tools and potential therapeutics. Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like G proteins that potently inhibit high voltage-activated (HVA) Ca (Ca1/Ca2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary Caβ-subunits. To investigate latent Caβ-independent components of inhibition, we coexpressed each RGK individually with Ca1 (Ca1.2/Ca1.3) or Ca2 (Ca2.1/Ca2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β or a mutant version (β) that does not bind RGKs. All four RGKs strongly inhibited Ca1/Ca2 channels reconstituted with WT β By contrast, when channels were reconstituted with β, Rem inhibited only Ca1.2, Rad selectively inhibited Ca1.2 and Ca2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT Caβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted Ca1.2 while Rad[R208A/L235A] inhibited Ca1.2/Ca2.2 but not Ca1.3/Ca2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous Ca1.2 channels in ventricular cardiomyocytes and selectively blocked 25 and 62%, respectively, of HVA currents in somatosensory neurons of the dorsal root ganglion, corresponding to their distinctive selectivity for Ca1.2 and Ca1.2/Ca2.2 channels. Thus, we have exploited latent β-binding-independent Rem and Rad inhibition of specific Ca1/Ca2 channels to develop selective GECCIs with properties unmatched by current small-molecule Ca channel blockers.