Rem, a member of the RGK GTPases, inhibits recombinant Ca_V1.2 channels using multiple mechanisms that require distinct conformations of the GTPase

Abstract

Rad/Rem/Gem/Kir (RGK) GTPases potently inhibit Ca V 1 and Ca V 2 (Ca V 1-2) channels, a paradigm of ion channel regulation by monomeric G-proteins with significant physiological ramifications and potential biotechnology applications. The mechanism(s) underlying how RGK proteins inhibit I Ca is unknown, and it is unclear how key structural and regulatory properties of these GTPases (such as the role of GTP binding to the nucleotide binding domain (NBD), and the C-terminus which contains a membrane-targeting motif) feature in this effect. Here, we show that Rem inhibits Ca V 1.2 channels by three independent mechanisms that rely on distinct configurations of the GTPase: (1) a reduction in surface density of channels is accomplished by enhancing dynamin-dependent endocytosis, (2)

Direct inhibition of P/Q-type voltage-gated Ca 2+ channels by Gem does not require a direct Gem/Ca v β interaction

“…The Ca v β-priming model proposed here for Gem may also be applicable to Rem and Rem2, because they both have been shown to inhibit surface Ca 2+ channels (18,19,21,25,31). This model remains speculative, and many questions remain to be answered, including the locations of the proposed anchoring site and inhibitory binding site for Gem in Ca v α 1 .…”

“…Thus, a prevalent hypothesis is that RGK proteins need to bind Ca v β to exert their inhibitory effect (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)31). To test this hypothesis, we abolished Gem/β 3 interaction by simultaneously mutating to alanine three residues on Gem and three residues on β 3 that have been shown to be critical for this interaction (34).…”

“…Inhibition of surface HVA Ca 2+ channels has also been observed for two other RGK proteins. Thus, Rem2 inhibits endogenous surface N-type channels in sympathetic and dorsal-root ganglion neurons (18), Rem inhibits surface L-type channels expressed in pancreatic β-cells (19,21) and HEK 293 cells (31), and rapid translocation of a recombinant Rem derivative acutely inhibits L-and N-type channels expressed in tsA201 cells (25).…”

“…Two modes of action have been reported: (i) RGK proteins reduce the number of HVA Ca 2+ channels on the cell surface, either by interfering with channel trafficking to the plasma membrane or increasing endocytosis of surface channels (14,16,17,20,24,31,34,35), and (ii) RGK proteins inhibit channels already on the plasma membrane (18,19,21,25,31). The molecular mechanisms of either mode of action are unknown, but because of the central role of Ca v β in HVA Ca 2+ -channel trafficking and gating, it is widely assumed that both forms of inhibition rely on the RGK/Ca v β interaction (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)31). This key hypothesis, however, has not been tested.…”

“…Recently, members of the Rem, Rem2, Rad, and Gem/Kir (RGK) family of Ras-related monomeric small GTP-binding proteins, which are known to regulate cytoskeleton remodeling through the Rho/Rho kinase signaling cascade (review in ref. 13), have emerged as the most potent protein inhibitors of HVA Ca 2+ channels (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). RGK proteins are present in many tissues and cells where HVA Ca 2+ channels are expressed, including the brain and cardiac, skeletal, and smooth muscles (review in ref.…”