The monomeric G proteins AGS1 and Rhes selectively influence Galphai-dependent signaling to modulate N-type (CaV2.2) calcium channels.

Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing Galphai and Rhes selectively influencing Galphas. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca(2+) channels (Ca(V)2.2) by selectively altering Galphai-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing Ca(V)2.2 and Galphai- or Galphas-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of Ca(V)2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through Galphai-coupled receptors without reducing channel inhibition through a Galphas-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gbetagamma-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on Ca(V)2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gbetagamma signaling and both attenuate receptor-initiated signaling by the Gbetagamma subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca(2+) influx through Ca(V)2.2 channels under more physiological conditions and thereby influence Ca(2+)-dependent events such as neurosecretion.

Neurokinin 1 receptors trigger overlapping stimulation and inhibition of CaV2.3 (R-type) calcium channels.

Neurokinin (NK) 1 receptors and CaV2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of CaV2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of CaV2.3. In particular, weak activation of these receptors evokes mainly stimulation of CaV2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by CaV2.3, L-type (CaV1.3), N-type (CaV2.2), and P/Q-type (CaV2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of CaV2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either Galpha(q/11) (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-beta1) or Gbeta gamma subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering Gbeta gamma, whereas a slow component of inhibition was reduced by buffering Galpha(q/11). When both Gbeta gamma and Galpha(q/11) were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of CaV2.3. We also report that NK1 receptors accelerate the inactivation kinetics of CaV2.3 currents. Altogether, our results indicate that NK1 receptors modulate CaV2.3 using three different signaling mechanisms: a fast inhibition mediated by Gbeta gamma, a slow inhibition mediated by Galpha(q/11), and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.

Muscarinic modulation of Cav2.3 (R-type) calcium channels is antagonized by RGS3 and RGS3T.

Ca(2+) influx through voltage-gated R-type (Ca(V)2.3) Ca(2+) channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that Ca(V)2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic ACh receptors. We previously demonstrated that muscarinic stimulation of Ca(V)2.3 is blocked by regulator of G protein signaling (RGS) 2. Here we investigated whether muscarinic inhibition of Ca(V)2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy ( approximately 380 residue) amino-terminal domain of uncertain physiological function. Ca(V)2.3, M(2) muscarinic ACh receptors (M(2)R), and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of Ca(V)2.3 was quantified using whole cell patch-clamp recordings. Full-length RGS3, RGS3T, and the core domain of RGS3 were equally effective in antagonizing inhibition of Ca(V)2.3 through M(2)R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type Ca(2+) channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-enhanced green fluorescent protein fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T, and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins, and other signaling proteins.

Reconstituted slow muscarinic inhibition of neuronal (Ca(v)1.2c) L-type Ca2+ channels.

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.