Abstract
Genetically encoded inhibitors for voltage-dependent Ca(2+) (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) Ca(2+) (Ca(V)1/Ca(V)2 family) channels, but their nonselectivity limits their potential applications. We hypothesized that nonselectivity of RGK inhibition derives from their binding to auxiliary Ca(V)β-subunits. To investigate latent Ca(V)β-independent components of inhibition, we coexpressed each RGK individually with Ca(V)1 (Ca(V)1.2/Ca(V)1.3) or Ca(V)2 (Ca(V)2.1/Ca(V)2.2) 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(V)1/Ca(V)2 channels reconstituted with WT β(2a) By contrast, when channels were reconstituted with β(2a,TM), Rem inhibited only Ca(V)1.2, Rad selectively inhibited Ca(V)1.2 and Ca(V)2.2, while Gem and Rem2 were ineffective. We generated mutant RGKs (Rem[R200A/L227A] and Rad[R208A/L235A]) unable to bind WT Ca(V)β, as confirmed by fluorescence resonance energy transfer. Rem[R200A/L227A] selectively blocked reconstituted Ca(V)1.2 while Rad[R208A/L235A] inhibited Ca(V)1.2/Ca(V)2.2 but not Ca(V)1.3/Ca(V)2.1. Rem[R200A/L227A] and Rad[R208A/L235A] both suppressed endogenous Ca(V)1.2 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(V)1.2 and Ca(V)1.2/Ca(V)2.2 channels. Thus, we have exploited latent β-binding-independent Rem and Rad inhibition of specific Ca(V)1/Ca(V)2 channels to develop selective GECCIs with properties unmatched by current small-molecule Ca(V) channel blockers.