Similar molecular determinants on Rem mediate two distinct modes of inhibition of CaV1.2 channels.
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 CaVchannels is important for perspectives on their (patho)physiological roles and could advance their development and use as genetically-encoded CaVchannel 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 α1Csubunit 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 α1CN-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.
a Department of Pharmacology & Molecular Signaling , Columbia University , New York , NY , USA.
Akil A
AA
Puckerin
b Department of Physiology & Cellular Biophysics , Columbia University , New York , NY , USA.
Donald D
DD
Chang
b Department of Physiology & Cellular Biophysics , Columbia University , New York , NY , USA.
Prakash
P
Subramanyam
a Department of Pharmacology & Molecular Signaling , Columbia University , New York , NY , USA.|b Department of Physiology & Cellular Biophysics , Columbia University , New York , NY , USA.
Henry M
HM
Colecraft
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006352:Heart Ventricles / Q000166:cytology; D006801:Humans; D008297:Male; D020559:Monomeric GTP-Binding Proteins / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000502:physiology; D017207:Rats, Sprague-Dawley
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); C108135:Rem protein, mouse; C490245:GEM protein, human; D020559:Monomeric GTP-Binding Proteins
CaV1.2; Gem; L-type calcium channel; RGK protein; Rem