Regulation of HCN channels by lipids and auxiliary subunits

超极化激活的环核苷酸门控（HCN）通道，像所有的离子通道一样，允许跨细胞膜传导离子。因此，离子通道与它们嵌入其中的脂质密切相互作用。然而，脂质景观是复杂和动态的，并随着细胞内的空间和时间而变化。此外，细胞膜的理化性质（头基，酰基尾不饱和度，疏水厚度，曲率）可以通过直接和间接的相互作用帮助控制蛋白质的功能。然而，尽管如此，离子通道的脂质调节仍然在很大程度上没有表征。这是因为直到最近，解决离子通道的脂质调节的努力只能在脂质组成复杂、知之甚少且难以控制的细胞中进行。最近在成功纯化功能性HCN通道方面取得了重大突破，这将使我们能够通过将纯化的HCN蛋白质重组到确定组成的脂质体中来检查它们的脂质依赖性。我已经使用这种方法来发现新的细节，脂质调节纯化的人内向整流钾+（Kir）通道，以及同源特异性阴离子脂质依赖性纯化细菌电压门控钠（Nav）通道。将这种方法与其他生物物理和结构技术（计算对接、电生理记录、脂质结合测定等）相结合，将使我们能够发现HCN通道的新调节机制，并了解其潜在的分子基础。除了脂质，辅助蛋白KCNE2调节HCN表达，电压依赖性和门控动力学。然而，目前尚不清楚KCNE2如何与HCN通道形成复合物，以及它们的调节如何与Ih的其他修饰剂相互作用（例如：cAMP、TRIP8b和脂质）。我假设HCN通道是由脂质膜的理化性质调节的，并且它们的脂质和蛋白质调节剂之间存在串扰。具体而言：* 目的1：确定脂质如何与HCN通道相互作用以调节其功能。电生理学，计算和生物化学的方法将被用来评估磷酸肌醇调节HCN通道的分子细节。HCN通道的新型脂质调节剂将通过将纯化的人HCN 1重组到确定组成的膜中并通过基于荧光的通量测定和电生理学检查通道活性作为脂质环境的函数来检查。**目标二：确定辅助蛋白KCNE2如何调节HCN功能，并确定KCNE2与其他HCN通道调节剂之间是否存在串扰。计算，生物化学和电生理学方法的组合将使我们能够评估HCN和KCNE2亚基之间相互作用的分子细节。我们还将确定KCNE2和HCN通道的脂质调节剂之间是否存在串扰。

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.***