Differential Modulation of Ca_V2.3 Ca²⁺Channels by Gαq/11-Coupled Muscarinic Receptors

Abstract

Ca V 2.3 subunits are expressed in neuronal and neuroendocrine cells where they are believed to form native R-type Ca 2ϩ channels. Although R-type currents are involved in triggering neurotransmitter and hormone secretion, little is known about their modulation. Previous studies have shown that muscarinic acetylcholine receptors evoke both inhibition and stimulation of Ca V 2.3. Muscarinic inhibition of Ca V 2.3 is mediated by G␤␥ subunits, whereas stimulation is mediated by pertussis toxininsensitive G␣ subunits. In the present study, we compared modulation of Ca V 2.3 by the three G␣q/11-coupled muscarinic receptors (M1, M3, and M5). Our data indicate that these receptors trigger comparable stimulation of Ca V 2.3. The signaling pathway that mediates stimulation was meticulously analyzed for M1 receptors. Stimulation is blocked by neutralizing antibodies directed against G␣q/11, coexpression of the regulatory domain of protein kinase C␦ (PKC␦), preactivating PKC with phorbol ester, or pharmacological suppression of PKC with bisindolylmaleimide I. Stimulation of Ca V 2.3 is Ca 2ϩ -independent and insensitive to 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö 6976), a specific inhibitor of Ca 2ϩ -dependent PKC isozymes. These results indicate that muscarinic stimulation of Ca V 2.3 involves signaling by G␣q/11, diacylglycerol, and a Ca 2ϩ -independent PKC. In contrast to stimulation, the magnitude of Ca V 2.3 inhibition depended on receptor subtype, with M3 and M5 receptors producing much larger Ca V 2.3 inhibition than M1 receptors. Interestingly, muscarinic inhibition of Ca V 2.3 was notably enhanced during pharmacological suppression of PKC, suggesting the presence of cross-talk between G␤␥-mediated inhibition and PKC-mediated stimulation of R-type channels similar to that described previously for N-type channels.

Neurokinin 1 Receptors Trigger Overlapping Stimulation and Inhibition of CaV2.3 (R-Type) Calcium Channels

“…Both the onset and recovery from stimulation (in response to 1 nM NKA) exhibited slow kinetics (Fig. 2, A and C), consistent with our previous reports (Meza et al, 1999;Melliti et al, 2000;Bannister et al, 2004).…”

“…Several previous studies demonstrated that Ca V 2.3 and native R-type currents are modulated by signaling mechanisms linked to G protein-coupled receptors (Wu et al, 1998;Yu and Shinnick-Gallagher, 1998;Meza et al, 1999;Melliti et al, 2000;Sabatini and Svoboda, 2000;Bannister et al, 2004;Kohlmeier and Leonard, 2006;Tai et al, 2006). In our previous experiments, we showed that Ca V 2.3 is both inhibited and stimulated through G␣ q/11 -coupled muscarinic acetylcholine receptors (Meza et al, 1999;Melliti et al, 2000;Bannister et al, 2004).…”

“…Previous studies have shown that Ca V 2.3 is stimulated by muscarinic acetylcholine and metabotropic glutamate receptors acting through PKC (Stea et al, 1995;Meza et al, 1999;Melliti et al, 2000;Kamatchi et al, 2003;Bannister et al, 2004). As shown in Fig.…”

“…In our previous experiments, we showed that Ca V 2.3 is both inhibited and stimulated through G␣ q/11 -coupled muscarinic acetylcholine receptors (Meza et al, 1999;Melliti et al, 2000;Bannister et al, 2004). The neurokinin (NK) 1 receptor is a G␣ q/11 -coupled receptor with a well established role in nociception (Macdonald et al, 1996;Duffy, 2004).…”

Voltage-Gated R-Type Calcium Channel Inhibition via Humanμ-,δ-, andκ-opioid Receptors Is Voltage-Independently Mediated by GβγProtein Subunits

“…Despite the clear functional importance of Ca v 2.3 channels in pain pathways, the mechanisms by which they are modulated by GPCRs have not yet been adequately examined (Rittenhouse, 2014). It is known that the Ca v 2.3 channel is regulated by muscarinic (Bannister et al, 2004), dopamine , and GABA B (Berecki et al, 2014) receptors. From reconstitution in heterologous expression systems, it is also known that Ca v 2.3 activity is weakly modulated via m-and k-ORs (Ottolia et al, 1998;Simen and Miller, 1998) but not d-ORs; lack of Ca v 2.3 modulation via m-OR has also been reported (Bourinet et al, 1996).…”