Direct Inhibition of the Interaction between α-Interaction Domain and β-Interaction Domain of Voltage-dependent Ca2+ Channels by Gem

Abstract

The Ras-related small G-protein Gem regulates voltagedependent Ca 2؉ channels (VDCCs) through interaction with the ␤-subunit of the VDCC. This action of Gem is mediated by regulated ␣ 1 -subunit expression at the plasma membrane. In the present study, we examined the mechanism of the inhibition of VDCC activity by Gem. The ␤-interaction domain (BID) of the ␤-subunit, which specifically interacts with the ␣-interaction domain (AID) of the ␣ 1 -subunit, is shown to be essential for the interaction between Gem and ␤-subunits. In addition, the AID peptide inhibited interaction between Gem and ␤-subunits in a dose-dependent manner. GemS88N mutant, which has low binding affinity for guanine nucleotide, did not interact with ␤-subunits, allowing ␣ 1 -subunit expression at the plasma membrane. This inhibitory effect of wild-type Gem on VDCC activity was reduced in cells expressing GemS88N. The overexpression of wild-type Gem in pancreatic ␤-cell line MIN6 cells suppressed Ca 2؉ -triggered secretion, whereas overexpression of GemS88N induced Ca 2؉ -triggered secretion to control level. These results suggest that GTPase activity of Gem is required for the binding of Gem to BID that regulates VDCC activity through interaction with AID.Voltage-dependent Ca 2ϩ channels (VDCC(s)) 1 permit the entry of Ca 2ϩ into excitable cells, coupling membrane potential changes to biological activities including muscle contraction, hormone and neurotransmitter release, neuronal migration, and gene expression (1-4). Based on the pharmacological and electrophysiological properties, VDCCs have been grouped into five subclasses, T, L, N, P/Q, and R (5-7). The VDCCs are multisubunit proteins composed of ␣ 1 , ␤, ␣ 2 /␦, and ␥-subunits.The ␣ 1 -subunit includes the pore of the channel, the voltage sensor, and the binding sites of drugs and toxins that modulate channel activity. Although the biophysical diversity of native VDCCs is conferred by ␣ 1 -subunits, auxiliary subunits ␤, ␣ 2 /␦, and ␥ modulate channel properties including current amplitude, voltage dependence, and kinetics of activation and inactivation (8 -11). Ten ␣ 1 -subunit isoforms have been identified (4). Four ␤-subunit isoforms, ␤ 1 , ␤ 2 , ␤ 3 , and ␤ 4 have been identified (12). Knock-out mice lacking the ␤ 1 , ␤ 2a , or ␤ 4 -subunits demonstrate that the ␤-subunit is essential for formation and functional expression of VDCCs (13-15).Coexpression of a ␤-subunit with an ␣ 1 -subunit results in an increase of peak current amplitude and an increase in the number of binding sites for drugs and toxins, acceleration of activation and inactivation kinetics, and an increase the number of ␣ 1 -subunits in plasma membrane (16 -19). The effects of ␤-subunits are suggested to be mediated by interaction with the intracellular I-II loop of ␣ 1 -subunits (20 -22). The I-II loop of the ␣ 1 -subunit includes the endoplasmic reticulum retention signal and inhibits trafficking of ␣ 1 -subunits to the plasma membrane. The interaction of the loop with the ␤-subunit masks the retention signal in t...

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

“…RGK-mediated Ca V β sequestration would result in the retention of newly synthesized Ca V α 1 subunits in an intracellular compartment [52], resulting in a chronic reduction in surface expressed Ca 2+ channels [74]. Subsequent studies have supported this observation [18,44,75], and extended it to demonstrate inhibition of epitope-tagged Ca V α 1 trafficking by co-expressed Rad, Rem and Rem2, as detected by immunofluorescence microscopy [18,41,43]. The finding that RGK protein localization is controlled in part through their association with calmodulin (CaM) and 14-3-3 proteins [41,43,44,46,47], prompted Beguin and colleagues to extend the sequestration regulatory model.…”

“…A key feature of the sequestration model is the notion that RGK proteins inhibit Ca V α:Ca V β association [52,75]. All Ca V β subunits share a conserved domain structure with three variable regions separated by conserved SH3-like and GK-like domains [77].…”

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

“…Studies are ongoing to examine whether regulation of these lipid second messengers provides a novel mechanism for controlling Rem-dependent Ca 2ϩ channel inhibition. Since Rem directly binds to a variety of accessory Ca V ␤ subunits (9), and a number of studies suggest that this interaction is required for the regulation of functional Ca 2ϩ channels at the plasma membrane (8,9,11,16), we examined whether Rem localization would be altered by co-expression of either Ca V 1.2 or Ca V ␤ subunits or in the presence of a functional Ca V 1.2-␤2a Ca 2ϩ channel. However, a similar fluorescence pattern was seen whether Ca V ␣ and/or Ca V ␤ subunits were present or absent in tsA201 cells (Fig.…”

“…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 ␤ association still appears critical (16,17).…”

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

“…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).…”