Structural and functional studies of the human TRPM4 and TRPM5 channels

项目总结 从心脏流向大脑的血液受到严格控制，以保护脆弱的脑组织，因为 血液流动会导致许多心血管疾病和脑损伤。TRPM4是主要的 细胞内调节脑动脉血管平滑肌细胞内血流的因子 压力会发生变化。TRPM4的突变或功能障碍与许多心血管疾病有关， 包括中风和Brugada综合征。TRPM4及其最接近的同系物Trpm5被钙激活， 非选择性、电压门控离子通道。Trpm5在胰岛β细胞中高表达，并且 Trpm5功能障碍或突变与II型糖尿病和肥胖症有关。此外，TRPM4和 味蕾细胞中的Trpm5在味觉信号中起着重要的作用，这两个通道的丢失都被取消了 察觉苦味、甜味或鲜味刺激的能力。总而言之，TRPM4和TrPM5的范围很广 在生理学和病理生理学中的作用。 TRPM4和Trpm5都属于黑素样瞬时受体潜能(TRPM)亚家族。 Trp超家族，是目前仅有的两个不透钙的成员。缺乏一个积极的规范 带电的电压敏感区域使TRPM4和TrPM5如何感知电压成为一个谜。尽管 TRPM4和M5有45%的氨基酸同源性，具有不同的功能和药理特性 动力学术语和对药物的敏感性。武田加州公司之间已经建立了合作关系。 和我们的实验室研究Trpm5在糖尿病治疗中的重要作用。特别是高亲和力药物 针对武田提供的Trpm5和潜在的未来药物开发，加强了我们关于 研究这两个经络的药理作用。目前，我们还不清楚，在分子细节上， 通道是以电压依赖的方式激活的，它们是如何被小分子调制的 在特定位置与它们结合，它们如何被各种药物区分，或者它们的通道如何发挥作用 受钙调蛋白等其他蛋白质的调节。 在成功解决了第一个关闭状态下的人类TRPM4结构的基础上，我们建议继续 TRPM4和Trpm5的冷冻-EM研究及其药理作用 电生理学实验和与武田的合作。这项提案的结果将定义 这些离子通道的电压依赖门控活性的分子基础，用于配体识别，以及 用于调制器的动作。这些进展反过来将为开发新的治疗方法提供基础。 抗心血管疾病和糖尿病的药物以及更深入地了解 电压门控TRPM家族成员。

PROJECT SUMMARY Blood flow from the heart to the brain is strictly regulated to protect the delicate brain tissue, because improper blood flow can give rise to numerous cardiovascular diseases and brain injuries. TRPM4 is one of the major actors regulating blood flow in the vascular smooth muscle cells in the cerebral arteries when intracellular pressure changes. Mutation or dysfunction of TRPM4 is linked to numerous cardiovascular diseases, including stroke and Brugada syndrome. TRPM4 and its closest homolog, TRPM5, are Ca2+-activated, nonselective, voltage-gated ion channels. TRPM5 is highly expressed in pancreatic beta cells, and dysfunction or mutation in TRPM5 is associated in type II diabetes and obesity. In addition, TRPM4 and TRPM5 in the taste bud cells play an important role in taste signaling, and loss of both channels abolishes the ability to detect bitter, sweet, or umami stimuli. Taken together, TRPM4 and TRPM5 have a wide range of roles in physiology and pathophysiology. Both TRPM4 and TRPM5 belong to the TRPM (melastatin-like transient receptor potential) subfamily of the TRP superfamily, and they are the only two members impermeable to Ca2+. The lack of a canonical positively charged voltage-sensing domain makes a mystery of how TRPM4 and TRPM5 sense voltage. Despite sharing 45% amino acid identity, TRPM4 and M5 have distinct functional and pharmacological properties in terms of kinetics and sensitivities to drugs. A collaboration has been built between Takeda California, Inc. and our lab to study the important role of TRPM5 in treatment of diabetes. The high-affinity drugs specifically targeting TRPM5 provided by Takeda and the potential future drug development strengthen our proposal on studying the pharmacology of these two channels. At present, we do not understand, in molecular detail, how the channels are activated in a voltage-dependent manner, how they are modulated by small molecules binding to them at specific sites, how they are distinguished by various drugs, or how their channel functions are modulated by other proteins such as calmodulin. Building on the success of solving the first human TRPM4 structure in closed state, we propose to continue the cryo-EM studies of TRPM4 and TRPM5 and their pharmacology, combined with complementary electrophysiology experiments and collaboration with Takeda. The outcome of this proposal will define the molecular basis for the voltage-dependent gating activity of these ion channels, for ligand recognition, and for the action of modulators. These advances, in turn, will provide a foundation for developing new therapeutic agents against cardiovascular diseases and diabetes and for a deeper understanding of the function of the voltage-gated TRPM family members.