Regulation of HCN channels by lipids and auxiliary subunits

超极化激活的环核苷酸门控（HCN）通道，像所有离子通道一样，允许离子穿过细胞膜传导。因此，离子通道与它们所嵌入的脂质密切相互作用。然而，脂质景观是复杂和动态的，并随着时间和空间的变化而变化。此外，细胞膜的理化性质（头群、酰基尾不饱和程度、疏水厚度、曲率）可能通过直接和间接的相互作用来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在很大程度上仍未被表征。这是因为直到最近，解决离子通道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蛋白重组成确定组成的脂质体来检查它们的脂依赖性。我已经使用这种方法揭示了纯化的人类内向整流K+ (Kir)通道的脂质调节的新细节，以及纯化的细菌电压门控钠（Nav）通道中同源特异性阴离子脂质依赖性。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通道调节的新机制，并了解其潜在的分子基础。除了脂质外，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其他Ih修饰因子相互作用。cAMP， TRIP8b和脂质)。我假设HCN通道受脂质膜的物理化学性质调节，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、计算和生化方法将用于评估磷酸肌苷调控HCN通道的分子细节。新的HCN通道脂质调节剂将通过将纯化的人HCN1重组到定义成分的膜中，并通过基于荧光通量测定和电生理学检测通道活性作为脂质环境的功能来检测。目的2：确定辅助蛋白KCNE2如何调节HCN功能，并确定KCNE2与HCN通道的其他调节因子之间是否存在串扰。计算、生化和电生理方法的结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通道的脂质调节因子之间是否存在串扰。

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.