RGS2 blocks slow muscarinic inhibition of N-type Ca(2+) channels reconstituted in a human cell line

• Published in: The Journal of physiology 2001-04, Vol.532 (Pt 2), p.337-347
• Main Authors: Melliti, K, Meza, U, Adams, B A
• Format: Article
• Language: English
• Subjects: Calcium Channels, N-Type - drug effects; Calcium Channels, N-Type - metabolism; Carbachol - antagonists & inhibitors; Carbachol - pharmacology; Cell Line; Electrophysiology; GTP-Binding Proteins - physiology; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Humans; Muscarinic Agonists - pharmacology; Pertussis Toxin; RGS Proteins - physiology; Staurosporine - pharmacology; Time Factors; Virulence Factors, Bordetella - pharmacology
• ISSN: 0022-3751
• DOI: 10.1111/j.1469-7793.2001.0337f.x

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.