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 expr essed in neuronal, atrial and endocrine tissues and play key roles in gener ating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate a nd modulating hormone release. They are activated directly by G(beta gamma) subunits released in response to G(i/o)-coupled receptor stimulation. Howe ver, it is not clear to what extent this process can be dynamically regulat ed by other cellular signalling systems, In this study we have explored pat hways 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 activati on of M-1 and M-3 receptors. This contrasts with the simple stimulation see n through activation Of M-2 and M-4 receptors. 3. Channel stimulation via M-1 but not M-3 receptors was sensitive to pertu ssis toxin whereas channel inhibition through both M-1 and M-3 receptors wa s insensitive. In contrast overexpression of the C-terminus of phospholipas e C beta1 or a G(q/11)-specific regulator of G protein signalling (RGS2) es sentially 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 th e inactive phorbol ester 4 alpha phorbol. Inhibition of the current by a sy nthetic analogue of diacylglycerol effectively occluded any further inhibit ion (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, 0-66 9 nm). 6. The expression of endogenous protein kinase C (PKC) isoforms in HEK293 c ells was examined by ininiunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated th e 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 ch annel stimulation by G(i/o)coupled receptors. 8. Our data support a role for a Ca2+ -independent PKC isoform in dynamic c hannel regulation, such that channel activity can be profoundly reduced by M-1 and M-3 muscarinic receptor stimulation.