Similar molecular determinants on Rem mediate two distinct modes of inhibition of Ca V 1.2 channels

Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (Ca 1/Ca 2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit Ca channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded Ca channel blockers. We previously reported that Rem can block surface Ca 1.2 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 subunit N-terminus (α -binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block Ca 1.2. Rem molecular determinants required for BBD Ca 1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and Ca β, respectively. However, Rem determinants for ABD Ca 1.2 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 1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α 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 1/Ca 2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of Ca 1.2 inhibition.