RG82 blocks slow muscarinic inhibition of N-type Ca2+ channels reconstituted in a human cell line

1. Native N-type Ca2+ channels undergo sustained inhibition through a slowl y activating pathway linked to M1 muscarinic acetylcholine receptors and G alphaq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N-t ype channels in HEK293 cells (a human embryonic kidney cell line) by coexpr essing cloned alpha 1B (Ca(v)2.2) Ca2+ channel subunits and M1, receptors. Expressed Ca2+ currents were recorded using standard whole-cell, ruptured-p atch techniques. 2. Rapid application of carbachol produced two kinetically distinct compone nts of Ca2+ 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 s taurosporine. 3. The fast component of inhibition was selectively blocked by the G beta g amma -binding region of beta -adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by G beta gamma released from G alphaq/11. 4. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with G alpha q/11 proteins. RGS2 also attenuated channel inhibition produced by intracel lular dialysis with non-hydrolysable GTP gammaX. Together these results sug gest that RGS2 selectively blocked slow inhibition by functioning as an eff ector antagonist, rather than as a GTPase-accelerating protein (GAP). 5. These experiments demonstrate that slow muscarinic inhibition of N-type Ca2+ 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 physiologica l regulator of the slow muscarinic pathway.