Regulation of HCN channels by lipids and auxiliary subunits

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Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, like all ion channels, allow for the conduction of ions across the cell membrane. Thus, ion channels intimately interact with the lipids in which they are embedded. However, the lipid landscape is complex and dynamic and varies with space and time within a cell. Also, the physiochemical properties of the cell membrane (headgroup, degree of acyl tail unsaturation, hydrophobic thickness, curvature) may help control the function of proteins through direct and indirect interactions. However, despite this, lipid regulation of ion channels remains largely uncharacterized. This is because until recently, efforts to address lipid regulation of ion channels could be only performed in cells where lipid composition is complex, poorly understood and difficult to control. Critical breakthroughs in successfully purifying functional HCN channels have been recently made, which will enable us to examine their lipid dependence by reconstituting purified HCN proteins into liposomes of defined composition. I have used this approach to uncover novel details of lipid regulation of purified human inward rectifier K+ (Kir) channels as well as homologue specific anionic lipid dependence in purified bacterial voltage-gated sodium (Nav) channels. Combining this approach with other biophysical and structural techniques (computational docking, electrophysiological recordings, lipid binding assays, etc.) will enable us to uncover novel mechanisms of regulation in HCN channels and understand their underlying molecular basis. In addition to lipids, the auxiliary protein KCNE2 regulates HCN expression, voltage-dependence, and gating kinetics. Yet, it remains unclear how KCNE2 forms a complex with HCN channels and how their regulation interacts with other modifiers of Ih (eg. cAMP, TRIP8b and lipids). I hypothesize that HCN channels are regulated by the physiochemical properties of lipid membrane, and that there is cross-talk between their lipid and protein modulators. Specifically: Aim 1: To determine how lipids interact with HCN channels to regulate their function. Electrophysiological, computational and biochemical approaches will be used to assess the molecular details of phosphoinositide regulation of HCN channels. Novel lipid regulators of HCN channels will be examined by reconstituting purified human HCN1 into membranes of defined composition and examining channel activity as a function of the lipid environment by fluorescence based flux assays and electrophysiology. Aim 2: To determine how the auxiliary protein KCNE2 regulates HCN function and determine if there is cross-talk between KCNE2 and other regulators of HCN channels. A combination of computational, biochemical, and electrophysiological approaches will enable us to assess the molecular details of interactions between HCN and KCNE2 subunits. We will also determine if there is cross-talk between KCNE2 and lipid regulators of HCN channels.