Regulation of a G protein‐gated inwardly rectifying K+ channel by a Ca2+‐independent protein kinase C

Abstract

Members of the Kir3.0 family of inwardly rectifying K+ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by Gβγ subunits released in response to Gi/o-coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the Gq/11-coupled M1 and M3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal-type channels stably expressed in the human embryonic kidney cell line HEK293. We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M1 and M3 receptors. This contrasts with the simple stimulation seen through activation of M2 and M4 receptors. Channel stimulation via M1 but not M3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M1 and M3 receptors was insensitive. In contrast over-expression of the C-terminus of phospholipase Cβ1 or a Gq/11-specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. The inhibitory effects of M1 and M3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor. The receptor-mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0–669 nm). The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε. We also demonstrate that activation of such a pathway via both receptor-mediated and receptor-independent means profoundly attenuated subsequent channel stimulation by Gi/o-coupled receptors. Our data support a role for a Ca2+-independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M1 and M3 muscarinic receptor stimulation.