Ion permeation, lipid flipping, and membrane remodeling by TMEM16 proteins

项目摘要/摘要 钙激活氯离子通道(CaCC)和其他TMEM16家族成员形成离子通道和/或 帮助协调大量细胞过程的脂类扰乱酶。人类表达了10种不同的 标记为TMEM16A-K(跳过I)的Paralog在全身表达，它们有助于 包括血液凝固、关节炎症信号抑制、疼痛控制等现象 通过伤害性神经元，以及调节多个大脑区域的神经元兴奋性--仅举一个例子 一些。这个家庭如何参与这么多不同的生理过程仍然是一个耐人寻味的问题 问题。创始成员(TMEM16A)于2008年由3个实验室(包括JAN实验室)克隆，使其 有可能阐明上面列出的生物角色，但也带来了剖析生物物理的能力 这些蛋白质的性质。在接下来的几年里，Jan实验室使用了突变筛选， 电生理学和小分子筛选以揭示离子传导、脂质扰乱和门控 除了解决高分辨率低温电磁结构外，TMEM16A和F的性质(与 TMEM16A(一种氯离子通道)和TMEM16F(一种双扰码/离子通道)的结构。 与此同时，格雷布实验室是第一个以原子细节展示nhTMEM16(真菌扰乱酶)如何翻转的实验室 通过在膜中诱导大范围的变形来使双层变薄，从而使亲水树林附近的双层变薄 艾滋病极地小组从一张传单传到另一张传单。尽管取得了这些进展，但根本性的问题 关于这些蛋白质的功能，我们打算在这里回答。第一，磷脂酰丝氨酸(PS) 通过TMEM16F暴露于质膜外叶是启动的关键信号事件 血小板依赖的凝血和微泡(MV)的产生；然而，还没有人证明 在生理条件下，TMEM16在原子水平上翻转带负电荷的PS分子，即脂质 人们对TMEM16s的特异性知之甚少，有人认为扰乱酶也可能 除了Grabe实验室揭示的那种模式外，还可以通过一种“出槽”模式来完成脂肪翻转。 其次，我们假设，在氯离子中，氯离子的传导是通过与膜屏蔽的专用孔道进行的。 选择性CACC，但尽管存在许多TMEM16A结构，这还没有显示出来。我们也 假设扰乱酶表现出依赖于脂质的选择性，因为离子与脂质在 蛋白质-膜界面。总之，我们的研究将揭示TMEM16如何 家庭成员会出现一系列不同的生物现象。

Project Summary/Abstract Calcium activated Chloride Channels (CaCCs) and other TMEM16 family members form ion channels and/or lipid scramblases that help orchestrate a large number of cellular processes. Humans express 10 different paralogs labeled TMEM16A-K (skipping I) that are expressed throughout the body, and they aid in diverse phenomena including coagulation of the blood, suppression of inflammatory signals in the joints, control of pain through nociceptive neurons, and modulating neuronal excitability in multiple brain regions – just to name a few. How this family can be involved in so many different physiological processes remains an intriguing open question. The founding member (TMEM16A) was cloned by 3 labs (including the Jan lab) in 2008 making it possible to elucidate the biological roles listed above, but also ushering in the ability to dissect the biophysical properties of these proteins. In the following years, the Jan lab employed mutagenesis screens, electrophysiology, and small molecule screening to uncover the ion conduction, lipid scrambling, and gating properties of TMEM16A and F in addition to solving high resolution cryo-EM structures (in collaboration with the Cheng lab) of TMEM16A (a Cl- channel) and structures of TMEM16F (a dual scramblase/ion channel). Meanwhile, the Grabe lab was the first to show in atomic detail how nhTMEM16 (a fungal scramblase) flips lipids by inducing large-scale deformations in the membrane that thin the bilayer near a hydrophilic grove that aids polar headgroups passing from one leaflet to the other. Despite these advances, fundamental questions about the function of these proteins remain that we intend to answer here. First, phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane via TMEM16F is the key signaling event that initiates platelet-dependent coagulation and microvesicle (MV) production; however, no one has demonstrated how a TMEM16 flips a negatively charged PS molecule at the atomic level under physiological conditions, the lipid specificity of TMEM16s is poorly understood, and it has been suggested that scramblases may also accomplish lipid flipping via an “out of the groove” mode in addition to the one revealed by the Grabe lab. Second, we hypothesize that Cl- conduction occurs via a dedicated pore shielded from the membrane in Cl- selective CaCC, but despite the existence of many TMEM16A structures, this has not been shown. We also hypothesize that scramblases exhibit selectivity that is lipid-dependent because ions co-permeate with lipids at the protein-membrane interface. Together, our studies will reveal basic mechanisms related to how TMEM16 family members carry out a diverse set of biological phenomena.