Modulation of cyclic nucleotide-regulated HCN channels by PIP2 and receptors coupled to phospholipase C

Recent results indicate that phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2), directly enhance the opening of hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels by shifting their activation gating to more positive voltages. This contrasts with the action of phosphoinositides to inhibit the opening of the related cyclic nucleotide-gated (CNG) channels involved in sensory signaling. We both review previous studies and present new experiments that investigate whether HCN channels may be regulated by dynamic changes in PI(4,5)P-2 levels caused by the receptor-mediated activation of phospholipase C (PLC). We coexpressed HCN1 or HCN2 channels in Xenopus oocytes with the PLC-coupled bradykinin BK2 receptor, the muscarinic M1 receptor, or the TrkA receptor. Activation of all three receptors produced a positive shift in HCN channel voltage gating, the opposite of the effect expected for PI(4,5)P-2 depletion. This action was not caused by alterations in cAMP as the effect was preserved in HCN mutant channels that fail to bind cAMP. The receptor effects were mediated by PLC activity, but did not depend on signaling through the downstream products of PI(4,5)P-2 hydrolysis: IP3 or diacylglycerol (DAG). Importantly, the modulatory effects on gating were blocked by inhibitors of phosphatidylinositol (PI) kinases, suggesting a role for increased PI(4,5)P-2 synthesis. Finally, we found that bradykinin exerted a similar PI kinase-dependent effect on the gating of native HCN channels in cardiac sinoatrial node cells, suggesting that this pathway may represent a novel, physiologically relevant mechanism for enhancing HCN channel function.