Rem Inhibits Cav1.2 Channels using both Beta-Binding Dependent and Independent Mechanisms

Abstract

Rad/Rem/Gem/Kir GTPases (RGKs) are the most potent known intracellular inhibitors of CaV1/CaV2 channels. Understanding their mechanism of action is critical not only for insights into their biological function but also may provide a blueprint for developing novel, useful protein CaV channel inhibitors. RGKs associate with auxiliary CaV beta subunits, but whether this interaction contributes to inhibition is elusive. We investigated the role of the RGK-beta interaction in Rem inhibition of reconstituted CaV1.2 channels, using a beta mutant (beta2aTM) selectively lacking binding to RGKs. Rem eliminated currents in wt beta2a-reconstituted channels (95% inhibition), but was significantly less potent (70% inhibition) in inhibiting beta2aTM-containing channels. Rem inhibits CaV1.2 channels using multiple mechanisms-decreasing channel number (N), open probability (Po), and gating charge (Qmax). The decreased N and Po (as demonstrated using a C1-domain-tagged Rem derivative that inducibly inhibits ICa,L when dynamically targeted to the membrane with PdBu, Figure) were eliminated in beta2aTM-reconstituted channels, indicating their beta-binding dependence. By contrast, reduced Qmax was retained in beta2aTM-reconstituted channels. The results shed new light on mechanisms of Rem inhibition of CaV1.2 channels and offer insights into rational development of novel bio-inspired CaV channel blockers.View Large Image | View Hi-Res Image | Download PowerPoint Slide