Structural and functional studies of the human TRPM4 and TRPM5 channels

项目摘要 从心脏到大脑的血液流动受到严格控制，以保护脆弱的脑组织，因为不适当的 血液流动可引起许多心血管疾病和脑损伤。TRPM 4是一个主要的 调节脑动脉中血管平滑肌细胞中血流的因子， 压力变化。TRPM 4的突变或功能障碍与许多心血管疾病有关， 包括中风和布鲁加达综合征TRPM 4及其最接近的同源物TRPM 5是Ca 2+激活的， 非选择性电压门控离子通道。TRPM 5在胰腺β细胞中高度表达， TRPM 5的功能障碍或突变与II型糖尿病和肥胖症有关。此外，TRPM 4和 味蕾细胞中的TRPM 5在味觉信号传导中起重要作用，两个通道的缺失会导致味觉信号传导的丧失。 感知苦味、甜味或鲜味刺激的能力。总的来说，TRPM 4和TRPM 5具有广泛的 在生理学和病理生理学中的作用。 TRPM 4和TRPM 5都属于TRPM（melastatin-样瞬时受体电位）亚家族， TRP超家族中的两个成员是唯一的不透Ca ~（2+）的成员。缺乏一个典型的积极 充电电压感应域使得TRPM 4和TRPM 5如何感应电压成为一个谜。尽管 TRPM 4和M5共享45%的氨基酸同一性，在免疫调节中具有不同的功能和药理学特性。 动力学和对药物的敏感性。Takeda加州公司与 本实验室研究TRPM 5在糖尿病治疗中的重要作用。高亲和力药物特别是 针对武田提供的TRPM 5和潜在的未来药物开发，我们提出了以下建议 研究这两个通道的药理学。目前，我们还不了解，在分子细节， 这些通道以电压依赖的方式被激活，它们如何被小分子调节， 在特定位点与它们结合，它们如何被各种药物区分，或者它们的通道如何起作用 由其他蛋白质如钙调蛋白调节。 在成功解决第一个人类TRPM 4结构封闭状态的基础上，我们建议继续 TRPM 4和TRPM 5的冷冻电镜研究及其药理学，结合互补的 电生理学实验和与武田的合作。该提案的结果将确定 这些离子通道的电压依赖性门控活性的分子基础，用于配体识别，以及 用于调节剂的作用。这些进展反过来将为开发新的治疗方法提供基础。 治疗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.