Phosphoinositides regulation of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels.

Abstract

Several ion channels are regulated by protein-lipid interactions. Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels specifically, have been shown to be regulated by phosphoinositides (PIPs). PIPs enhance HCN activation via a rightward shift in voltage-dependency and has importance applications in neuronal and cardiac function. Using computational and electrophysiological approaches, we aim to examine putative sites for HCN-PIP interactions. Computational docking and coarse-grained simulations indicate that PIP binding to HCN1 and HCN4 channels are not well coordinated, but rather occurs over a broad surface of charged residues primarily in the HCN-domain, S2 and S3 helices that can be loosely organized in 2 or 3 overlapping clusters. Thus, HCN-PIP interactions are more resembling of electrostatic interactions that occur in myristoylated alanine-rich C kinase substrate (MARCKS) proteins, than the specifically coordinated interactions that occur in pleckstrin homology domains (PH domains) or ion channels such as inward rectifier potassium (Kir) channels. Our results also indicate that phosphatidylinositol (PI) interactions with HCN1 are even lower affinity, explaining why unphosphorylated PI have no effect on HCN1 activation unlike phosphorylated PIPs. Experimental validation of putative binding sites are addressed by electrophysiological approaches.