Plasma Membrane Targeting Is Essential for Rem-mediated Ca2+ Channel Inhibition

Abstract

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca 2؉ channels and a known interacting partner for Ca 2؉ channel accessory ␤ subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca V ␤ association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membranelocalized ␤2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with ␤2a in vitro and ␤1b in vivo, suggesting that the C terminus does not directly participate in Ca V ␤ association. Despite demonstrated ␤1b binding, Rem-(1-265) was not capable of regulating a Ca V 1.2-␤1b channel complex, indicating that ␤ subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca 2؉ channel regulation, suggesting that ␤ binding and membrane localization are independent events required for channel inhibition.High voltage-activated Ca 2ϩ channels (Ca V 1 and Ca V 2 families) transduce electrical activity into increased intracellular calcium that mediates a diverse array of essential cellular processes, including hormone secretion, neurotransmitter release, and excitation-contraction coupling in muscle systems (1). The cardiac L-type Ca 2ϩ channel is a multiprotein complex consisting of the pore-forming Ca V 1.2 ␣ subunit and auxiliary subunits, including Ca V ␤ and ␣ 2 -␦ subunits (1). The Ca V ␣ subunit determines the ion selectivity and single channel conductance of the mature channel, whereas co-expression of Ca V ␤ or ␣ 2 ␦ facilitates cell surface trafficking of the ␣ 1 subunit, increases Ca 2ϩ current amplitude, and alters channel gating properties (1, 2). Ca V ␤ subunits are encoded by four genes (␤1-␤4), each subject to complex splicing (3). Ca V ␤2a, a ␤ isoform found in the heart, is subject to post-translational palmitoylation, which directs plasma membrane localization, whereas other ␤ isoforms are predominantly localized to the cytosol when not bound to Ca V ␣ 1 (3).Recently, members of the RGK 3 family of Ras-related GTPases, including Rem (4), Rem2 (5), Rad (6), and Gem/Kir (7), have been identified as potent regulators of HVA Ca 2ϩ channel function (8 -10). Although all RGK GTPases associate with Ca V ␤ subunits and prevent de novo expression of L-type I Ca (8 -10), the mechanism of RGK protein-mediated Ca 2ϩ channel inhibition remains controversial. It was originally hypothesized that RGK protein binding blocked Ca V ␣1/␤ association, leading to a reduction of functional channels at the cell surface (8,(11)(12)(13)(14). However, a series of recent studies suggests instead that the majority of RGK proteins inhibit the activity of the preassembled channel complex at the plasma membrane (10, 15, 16), although Ca V...

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…3). Ectopic expression of RGK proteins in variety of heterologous and endogenous cell models consistently demonstrates an almost complete inhibition of VDCC current, including L- [23,[52][53][54][55][70][71][72][73], P/Q- [52], and N- [49,52], but importantly not T-type channels which do not require accessory β-subunits for ionic current expression [55,69]. Recent studies in Xenopus oocytes demonstrate that this may be a dose-dependent effect [72], and recent work indicates that RGK-mediated Ca V β binding is essential for VDCC regulation [71].…”

“…RGK proteins do not contain canonical lipid modification motifs [48], though there is enrichment of these proteins at the plasma membrane, and individual proteins have been shown to be localized to the cytosol, nucleus, and with both the actin and microtubule networks [4,5,18,41,43,44,[46][47][48][49][50][51][52][53][54]. The carboxyl terminus is well conserved (Fig.…”

“…The carboxyl terminus is well conserved (Fig. 1) and has been shown to play a critical role in the function of all RGK GTPases [49,55], regulating subcellular distribution [4,5,53], and directing protein-protein interactions [45,53,55], to control both Ca 2+ channel activity [49,53,55] and cytoskeletal reorganization [41][42][43][44]46,47,52]. This region contains a cysteine residue within a highly conserved domain termed the C-7 motif (Fig.…”

“…This region contains a cysteine residue within a highly conserved domain termed the C-7 motif (Fig. 1 and [56]), however, despite the potential to serve as a site of lipidation, there is no evidence that the cysteine residue is modified, and deletion of the C-7 domain does not alter RGK function [8,48,53,55]. The question of how RGK GTPases localize to the plasma membrane was, at least partially, answered by the recent work of Heo and colleagues illustrating the importance of phosphatidylinositol phosphate (PIP) lipids in the membrane recruitment of RGKs, as well as other Ras-related GTPases containing polybasic tracts in their C-termini [57] (Figs.…”

“…1 and 3). Additional studies will be necessary to determine whether PIP lipid binding is required for RGK activity, although the recent analysis of Rem and Rem2 C-terminal truncation mutants suggests a strong correlation between PIP-lipid interaction, plasma membrane localization, and Rem/Rem2-dependent Ca 2+ channel regulation ( [53] and unpublished data). In addition to plasma membrane targeting through the C-terminus, three specific nuclear localization signals are well conserved in all RGK proteins [46,47].…”