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

Genetically encoded inhibitors for voltage-dependent Ca²⁺ (Ca_V) 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) Ca²⁺ (Ca_V1/Ca_V2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary Ca_Vβ-subunits. To investigate latent Ca_Vβ-independent components of inhibition, we coexpressed each RGK individually with Ca_V1 (Ca_V1.2/Ca_V1.3) or Ca_V2 (Ca_V2.1/Ca_V2.2) channels reconstituted in HEK293 cells with either wild-type (WT) β_2a or a mutant version (β_2a,TM) that does not bind RGKs. All four RGKs strongly inhibited Ca_V1/Ca_V2 channels reconstituted with WT β_2a By contrast, when channels were reconstituted with β_2a,TM, Rem inhibited only Ca_V1.2, Rad selectively inhibited Ca_V1.2 and Ca_V2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT Ca_Vβ, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted Ca_V1.2 while Rad[R208A/L235A] inhibited Ca_V1.2/Ca_V2.2 but not Ca_V1.3/Ca_V2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous Ca_V1.2 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 Ca_V1.2 and Ca_V1.2/Ca_V2.2 channels. Thus, we have exploited latent β-binding-independent Rem and Rad inhibition of specific Ca_V1/Ca_V2 channels to develop selective GECCIs with properties unmatched by current small-molecule Ca_V channel blockers.