RGS2 blocks slow muscarinic inhibition of N-type Ca2%2b channels reconstituted in a human cell line
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1. Native N-type Ca2%2b channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Gαq/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 α1B (Cav2.2) Ca2%2b channel subunits and M1 receptors. Expressed Ca2%2b currents were recorded using standard whole-cell, ruptured-patch techniques. 2. Rapid application of carbachol produced two kinetically distinct components of Ca2%2b 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 Gβγ-binding region of β-adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gβγ released from Gαq/11. 4. The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Gαq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non-hydrolysable GTPγS. 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 Ca2%2b 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 regulatory of the slow muscarinic pathway.
