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

Abstract

Rad/Rem/Rem2/Gem (RGK) proteins are Ras-like GTPases that potently inhibit all high-voltage-gated calcium (CaV1/CaV2) channels and are, thus, well-positioned to tune diverse physiological processes. Understanding how RGK proteins inhibit CaV channels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded CaV channel blockers. We previously reported that Rem can block surface CaV1.2 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 α1C subunit N-terminus (α1C-binding-dependent inhibition, or ABD). By contrast, Gem uses only the BBD mechanism to block CaV1.2. Rem molecular determinants required for BBD CaV1.2 inhibition are the distal C-terminus and the guanine nucleotide binding G-domain which interact with the plasma membrane and CaVβ, respectively. However, Rem determinants for ABD CaV1.2 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 CaV1.2 inhibition, but with different interaction partners. Rem distal C-terminus interacts with α1C 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 CaV1/CaV2 channels. The results reveal that similar molecular determinants on Rem are repurposed to initiate 2 independent mechanisms of CaV1.2 inhibition.

Designer genetically encoded voltage‐dependent calcium channel inhibitors inspired by RGK GTPases

“…2012; Puckerin et al . 2016, 2018). In the particular case of Rem inhibition of Ca V 1.2, the β‐binding‐independent mechanism of inhibition is mediated by an interaction of the Rem distal C‐terminus with the α 1C N‐terminus region just upstream of the first transmembrane spanning segment of the channel (Yang et al .…”

Defining the MO's of RGK proteins

“…Gem and Rem2 operate only through b-dependent mechanisms. 6,7 channels is far, far more complex than we first had thought.…”

“…8 In addition to defining the structural elements of Rem that are important for ABD inhibition of Ca V 1.2, Puckerin and colleagues showed that this form of inhibition is physiologically relevant in cardiomyocytes as adenoviral expression of the a 1C amino-terminus significantly reduced inhibition of native L-type currents by coexpressed Rem. 7 Since ABD inhibition has only been demonstrated to be a property of Ca V 1.2 channels so far, one can envision that specific therapeutic strategies based on the Rem-Ca V 1.2 interaction could be developed. 7,8 Still, more investigation of these fundamental mechanisms is necessary since it is now clear that RGK protein-mediated inhibition of Ca V Figure 1.…”

“…7 Since ABD inhibition has only been demonstrated to be a property of Ca V 1.2 channels so far, one can envision that specific therapeutic strategies based on the Rem-Ca V 1.2 interaction could be developed. 7,8 Still, more investigation of these fundamental mechanisms is necessary since it is now clear that RGK protein-mediated inhibition of Ca V Figure 1. Three in one: inhibition of L-type Ca V 1.2 channels by Rem.…”

“…7 To accomplish this goal, they employed 3-cube FRET and co-immunoprecipitations to demonstrate that the a 1C amino-terminus interacts with residues 265-285 in the distal carboxyl-terminus of Rem. ABD inhibition of L-type current was blunted in HEK293 cells expressing Rem constructs lacking this portion of the carboxyl-terminus in manner consistent with the aforementioned binding assays, but expression of the Rem carboxyl-terminus (residues 224-297) alone was insufficient for ABD inhibition.…”