Modulation of L-type Ca2+ Channels by Gβγ and Calmodulin via Interactions with N and C Termini of α1C

Abstract

Neuronal voltage-dependent Ca2؉ channels of the N (␣ 1B ) and P/Q (␣ 1A ) type are inhibited by neurotransmitters that activate G i/o G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of G␤␥ subunit of G proteins to the channel. Since cardiac and neuronal L-type (␣ 1C ) voltage-dependent Ca 2؉ channels are not modulated in this way, they are presumed to lack interaction with G␤␥. However, here we demonstrate that both G␤␥ and calmodulin directly bind to cytosolic N and C termini of the ␣ 1C subunit. Coexpression of G␤␥ reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca 2؉ , and is eliminated by EGTA. The N and C termini of ␣ 1C appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of G␤␥. Deletion of the N terminus profoundly impairs the Ca 2؉ /calmodulin-dependent inactivation. We propose that G␤␥ and calmodulin regulate the L-type Ca 2؉ channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of ␣ 1C .Voltage-dependent Ca 2ϩ channels (VDCCs) 1 are crucial for neuronal and muscular excitability (1). Mammalian VDCCs fall into several families distinguished by pharmacological and biophysical properties (L, N, P/Q, T, and R type) and the molecular identity of the main, poreϪforming subunit, ␣ 1 (2-4). The neuronal N-and P/Q-type channels, based on ␣ 1B and ␣ 1A , respectively, are crucial for neurotransmitter release (3). Ltype Ca 2ϩ channels containing the "cardiac-type" ␣ 1C subunit regulate contraction of cardiac and smooth muscle, and excitability and gene expression in the brain (2, 5, 6). The ␣ 1 subunits contain four homologous membrane domains numbered IϪIV and 5 large intracellular segments: N terminus (NT), C terminus (CT), and linkers between the domains (often called loops L 1 , L 2 , and L 3 ). There is also a large number of short intracellular linkers between transmembrane segments within each domain.Activation in all voltage-dependent channels is initiated by a voltage-driven shift in charged transmembrane elements (7). Nevertheless, the parts of the channel and the auxiliary subunits which are not exposed to the membrane electrical field may substantially modulate the gating (for reviews related to Ca 2ϩ channels, see Ref.3). In particular, VDCCs are strongly and specifically modulated by neurotransmitters acting via heterotrimeric G proteins, via actions on the cytosolic parts of the channel. Some of the modulations are mediated by G protein-triggered second messenger cascades, often via protein kinases A and C (PKA and PKC, respectively), others by a direct interaction with G protein subunits (1, 8 -14). Both PKC and PKA alter VDCC gating parameters acting via cytosolic parts of ␣ 1 or via the ancillary ␤ subunit (15-19).Neuronal VDCCs are usually inhibited by G protein-coupled rece...

Molecular determinants of Ca 2+ /calmodulin-dependent regulation of Ca v 2.1 channels

“…The functional roles of the CBD in CDI and CDF that we have detected by using the rat brain ␣ 1 2.1 (rbA isoform) may be less apparent with the human ␣ 1 2.1 isoform used in the previous work (6). Although the C-terminal regions, including the IQ-like domain and the CBD, are quite similar in the human and rat sequences, functionally significant differences may exist within these conserved sequences or in other parts of the channel that could influence channel modulation by CaM (21,22).…”

Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction

“…In combination, these results suggest that neither the cAMP-PKA pathway nor the phospholipase C-PKC pathway are involved directly in the modulation of spontaneous secretion by AD. Recently, Ivanina et al (2000) have demonstrated that both the Gbg subunit of G protein and calmodulin directly bind to the cytosolic N terminus and the C terminus of the a 1C subunit of L-type VDCCs, reducing the conductance of the channel. The inhibition occurs at basal cellular levels of Ca 2 þ , and is eliminated by EGTA.…”

“…The activation of A 1 AD receptors in cholinergic interneurons has been shown to reduce N-type Ca 2 þ currents via a membrane-delimited, G i/o class G-protein pathway (Song et al, 2000). Although the inhibitory action of Gbg protein seems to be selective for some types of Ca 2 þ channels, N-and P/Q-type VDCCs being the principal targets (Snutch & Reiner, 1992;Zhang et al, 1996;Jarvis & Zamponi, 2001), Ivanina et al (2000) have demonstrated that, at basal cellular levels of Ca 2 þ , G protein bg subunits have an inhibitory effect on L-type VDCCs in a voltage-independent, but calmodulin-dependent manner. Here we propose that CCPA inhibits spontaneous ACh secretion by a mechanism that involves Ca 2 þ -calmodulin, since treatment of preparations with the calmodulin antagonist W-7 and the intracellular Ca 2 þ chelator EGTA-AM eliminates the modulatory effect of CCPA.…”

G protein inhibition of CaV2 calcium channels

“…Two-out-of-thirty mutations (Arg 376 and Val 416 to alanine) significantly reduced the extent of voltage-dependent inhibition. Moreover, mutation of Arg 376 to phenylalanine increased inhibition, further implicating this residue in binding of Gβγ C terminal tails of Ca 2+ channels, [92][93][94] and Ca V β binding to I-II linker appears to play a role in Ca 2+ -dependent inactivation. 82 As discussed below, these regions of the channel are also intimately involved in voltage-dependent, Gβγ-mediated inhibition of Ca V 2 channels.…”