Similar molecular determinants on Rem mediate two distinct modes of inhibition of Ca_V1.2 channels

Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (Ca_V1/Ca_V2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit Ca_V channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded Ca_V channel blockers. We previously reported that Rem can block surface Ca_V1.2 channels in 2 independent ways that engage distinct components of the channel complex: (1) by binding auxiliary β subunits (β-binding-dependent inhibition, or BBD); and (2) by binding the pore-forming α_1C subunit N-terminus (α_1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block Ca_V1.2. Rem molecular determinants required for BBD Ca_V1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and Ca_Vβ, respectively. However, Rem determinants for ABD Ca_V1.2 inhibition are unknown. Here, combining fluorescence resonance energy transfer, electrophysiology, systematic truncations, and Rem/Gem chimeras we found that the same Rem distal C-terminus and G-domain also mediate ABD Ca_V1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α_1C N-terminus to anchor the G-domain which likely interacts with an as-yet-unidentified site. In contrast to some previous studies, neither the C-terminus of Rem nor Gem was sufficient to inhibit Ca_V1/Ca_V2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of Ca_V1.2 inhibition.