Reconstituted Slow Muscarinic Inhibition of Neuronal (Ca V1.2c) L-Type Ca 2+ Channels

Abstract

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 V1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. G αq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca V1.2c, whereas G αi/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 V1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against G αq/11. In contrast, inhibition was not reduced by coexpressing βARK1ct or G αt. These results indicate that slow inhibition required signaling by G αq/11, but not G βγ, subunits. Slow inhibition did not require Ca 2+ transients or Ca 2+ influx through Ca V1.2c 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 V1.2 (Ca V1.2a) and a splice-variant of the neuronal/neuroendocrine Ca V1.3 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.