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

1. 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 G(beta gamma) subunits released in response to G(i/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 G(q/11)-coupled M-1 and M-3 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. 2. We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M-1 and M-3 receptors. This contrasts with the simple stimulation seen through activation Of M-2 and M-4 receptors. 3. Channel stimulation via M-1 but not M-3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M-1 and M-3 receptors was insensitive. In contrast overexpression of the C-terminus of phospholipase C beta1 or a G(q/11)-specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4. The inhibitory effects of M-1 and M-3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4 alpha phorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor. 5. The receptor-mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0-669 nm). 6. The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by ininiunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation. of the novel PXC isoforms PXC delta and PKC epsilon. 7. We also demonstrate that activation of such a pathway via both receptor-mediated and receptor-independent means profoundly attenuated subsequent channel stimulation by G(i/o)coupled receptors. 8. Our data support a role for a Ca2+ -independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M-1 and M-3 muscarinic receptor stimulation.