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

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.
Competing Interests: The authors declare no conflict of interest.
(Copyright © 2018 the Author(s). Published by PNAS.)