Mechanisms of Disease associated with mechanically-activated Piezo ion channels

PIEZO1和PIEZO 2离子通道对我们的触觉和本体感觉以及检测 肺伸展和血管血流。到目前为止，已有8种不同的人类疾病与61种疾病有关 Piezos中的单点突变，其中许多与已知的生理上的 功能。虽然对于大多数突变来说，它们对Piezo功能的影响是未知的，但如此研究的少数突变 非常明显地影响压电失活，这本身并不是机械地理解的。这项应用的总体目标是全面描述目前已知的所有与人类疾病相关的机械激活的Piezo离子通道突变，并解决失活机制。我们的理论基础是，通过确定每个点突变的功能影响和了解Piezo失活的机制，我们采取了理解这些疾病所必需的两个前两步。我们的中心假设是，与人类疾病相关的Piezo单点突变影响膜表达、离子渗透或开放概率，而Piezo失活是由C末端细胞外域(CED)内的特定结构(残基/结构域)决定的。这一假设的科学前提是基于以下事实：i)被诊断为大肠息肉、脱水口腔细胞增多症、淋巴发育不良、溶血性贫血和远端关节畸形、马登-沃克综合征、戈登综合征、小眼炎的人类患者分别与Piezo1和Piezo2的突变有关，ii)失活是由CED和已知的功能性 突变、配体和电压对Piezos的调制，以及iii)我们自己的研究表明，人类 疾病相关的点突变改变了失活动力学，深刻改变了重复序列的转导 Piezos在机械振动过程中可能遇到的机械刺激，重复的肺伸展 呼吸，或心跳时脉动的血液流动。我们的具体目标将检验以下假设： 目的：确定61个单点突变对Piezo1和Piezo2功能的影响；AIM2：鉴定 失活的结构和分子机制。这项拟议的研究具有创新性，因为我们探索了61个与Piezo1和Piezo2疾病相关的人类单点突变的功能后果，几乎所有这些突变都在功能水平上尚未确定，还因为我们将确定失活机制及其结构相关性，这两者目前都是未知的。这项研究的意义是对与未知机制的人类疾病相关的Piezo点突变的功能效应进行全面的生物物理分析，并以失活的机制和结构探索为目标。这一知识将使我们深入了解这些疾病的潜在机制，并指导进一步的机制研究 探索，有效的诊断和疾病的治疗。

Piezo1 and Piezo2 ion channels are essential for our senses of touch and proprioception, and the detection of lung stretch and vascular blood flow. As of today, 8 distinct human diseases have been associated with 61 single-point mutations in Piezos, many of which are not obviously related to their known physiological functions. While for most mutations their effects on Piezo function are unknown, the few mutations studied thus far distinctly affect Piezo inactivation, which is itself not understood mechanistically. The overall objective of this application is a comprehensive functional characterization of all currently-known human disease-related mutations in mechanically-activated Piezo ion channels and solving the mechanism of inactivation. Our rationale is that by determining functional effects of each point-mutation and by knowing the mechanism of Piezo inactivation we take the two first steps necessary for understanding these diseases. Our central hypothesis is that single-point mutations in Piezos that have been associated with human diseases affect membrane expression, ion permeation, or open probability, and that Piezo inactivation is determined by specific structures (residues/domains) within the C-terminal-extracellular domain (CED). The scientific premise for this hypothesis is based on the facts, that i) human patients diagnosed with colorectal polyposis, dehydrated stomatocytosis, lymphatic dysplasia, hemolytic anemia, and distal arthrogryposis, Marden-Walker syndrome, Gordon syndrome, microphthalmia are associated with mutations in Piezo1 and Piezo2, respectively, that ii) inactivation is conferred by the CED and the known main target of functional modulation of Piezos by either mutations, ligands, and voltage, and iii) our own studies showing that human disease-related point-mutations that alter inactivation kinetics profoundly change transduction of repetitive mechanical stimuli, which Piezos likely encounter during mechanical vibrations, repetitive lung stretch during breathing, or pulsating blood flow upon heart beating. Our specific aims will test the following hypotheses: Aim1: Determine the effects of 61 single-point mutations on Piezo1 and Piezo2 function; Aim2: Identification of the structures and molecular mechanism of inactivation. The proposed research is innovative, because we explore the functional consequences of 61 human Piezo1 and Piezo2 disease-related single-point mutations, nearly all of which have remained uncharacterized on a functional level, and because we will identify the mechanism of inactivation and its structural correlates, both of which are currently unknown. The significance of this study is a comprehensive biophysical analysis of functional effects of Piezo point-mutations that have been associated with human diseases of unknown mechanisms, and the mechanistic and structural exploration of inactivation as their target. This knowledge will give deep insight into the mechanisms underlying these diseases and guide strategies for further mechanistic explorations, effective diagnosis and disease treatment.