Determinants of the voltage dependence of G protein modulation within calcium channel β subunits

CaVβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3) domain and a guanylate kinase-like (GK) domain with an intervening HOOK domain. We have shown in a previous study that, although Gβγ-mediated inhibitory modulation of CaV2.2 channels did not require the interaction of a CaVβ subunit with the CaVα1 subunit, when such interaction was prevented by a mutation in the α1 subunit, G protein modulation could not be removed by a large depolarization and showed voltage-independent properties (Leroy et al., J Neurosci 25:6984–6996, 2005). In this study, we have investigated the ability of mutant and truncated CaVβ subunits to support voltage-dependent G protein modulation in order to determine the minimal domain of the CaVβ subunit that is required for this process. We have coexpressed the CaVβ subunit constructs with CaV2.2 and α2δ-2, studied modulation by the activation of the dopamine D2 receptor, and also examined basal tonic modulation. Our main finding is that the CaVβ subunit GK domains, from either β1b or β2, are sufficient to restore voltage dependence to G protein modulation. We also found that the removal of the variable HOOK region from β2a promotes tonic voltage-dependent G protein modulation. We propose that the absence of the HOOK region enhances Gβγ binding affinity, leading to greater tonic modulation by basal levels of Gβγ. This tonic modulation requires the presence of an SH3 domain, as tonic modulation is not supported by any of the CaVβ subunit GK domains alone

Inhibition of native 5-HT3 receptor-evoked contractions in Guinea pig and mouse ileum by antimalarial drugs

Quinine, Chloroquine and mefloquine are commonly used to treat malaria; however with associated gastrointestinal (GI) side-effects. These drugs act as antagonists at recombinant 5-HT3 receptors and modulate gut peristalsis. These gastrointestinal side effects may be the result of antagonism at intestinal 5-HT3 receptors. Ileum from male C57BL/6 mice and guinea pigs was mounted longitudinally in organ baths. Concentration-response curves for 5-HT and the selective 5-HT3 agonist 2-Me-5-HT were obtained with 5-HT (pEC50=7.57±0.33, 12) more potent (P=0.004) than 2-Me-5-HT (pEC50=5.45±0.58, n=5) in mouse ileum. There was no difference in potency of 5-HT (pEC50=5.42±0.15, n=8) and 2-Me-5-HT (pIC50=5.01±0.55, n=11) in guinea pig ileum (P>0.05). Quinine, Chloroquine or mefloquine was applied for 10 min and inhibitions prior to submaximal agonist application. In mouse ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50=4.9±0.17, n=7; 4.76±0.14,n=5; 6.21±0.2, n=4, respectively) with mefloquine most potent (P<0.05). Quinine, chloroquine and mefloquine antagonised 2-me-5-HT-induced contractions (pIC50=6.35±0.11,n=8; 4.64±0.2, n=7; 5.11± 0.22, n=6, respectively) with quinine most potent (P<0.05). In guinea-pig ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50=5.02±0.15, n=6; 4.54±0.1, n=7; 5.32±0.13, n=5, respectively) and 2-me-5-HT-induced contractions (pIC50=4.62±0.25, n=5; 4.56±0.14, n=6; 5.67±0.12, n=4, respectively) with chloroquine least potent against 5-HT and mefloquine most potent against 2-me-5-HT (P<0.05). These results support previous studies identifying anti-malarial drugs as antagonists at recombinant 5-HT3 receptors and may also demonstrate the ability of these drugs to influence native 5-HT3 receptor-evoked contractile responses which may account for their associated GI side-effects

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classical transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx and cell responses. METHODS: We created trpc4-, trpc6- and trpc4/trpc6-gene deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies >80% of the muscarinic receptor-induced cation current or mI(CAT). The residual mI(CAT) depends on the expression of TRPC6 indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal mocytes the carbachol-induced membrane depolarizations are greatly diminished and the atropine sensitive contraction elicited by acetylcholine release from excitatory motor neurons is greatly reduced. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells, voltage-activated Ca(2+)-influx and contraction and thereby accelerate small intestinal motility in vivo