RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line.
1. Native N-type Ca(2+) channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Galphaq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N-type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned alpha1B (Ca(V)2.2) Ca(2+) channel subunits and M1 receptors. Expressed Ca(2+) currents were recorded using standard whole-cell, ruptured-patch techniques. 2. Rapid application of carbachol produced two kinetically distinct components of Ca(2+) channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5-40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. 3. The fast component of inhibition was selectively blocked by the Gbetagamma-binding region of beta-adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gbetagamma released from Galphaq/11. 4. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Galphaq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non-hydrolysable GTPgammaS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase-accelerating protein (GAP). 5. These experiments demonstrate that slow muscarinic inhibition of N-type Ca(2+) channels can be reconstituted in non-neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.
Melliti
K
K
Department of Biology, Utah State University, Logan 84322-5305, USA.
Meza
U
U
Adams
B A
BA
D020864:Calcium Channels, N-Type / Q000187:drug effects* / Q000378:metabolism*; D002217:Carbachol / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D002460:Cell Line; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology*; D037342:Pertussis Toxin; D020710:RGS Proteins / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D020864:Calcium Channels, N-Type; D018721:Muscarinic Agonists; D020710:RGS Proteins; C105345:Rgs2 protein, mouse; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019311:Staurosporine