Calcium Dynamics in Interstitial Cells of Cajal

项目摘要/摘要 外源性神经系统、肠神经、神经胶质细胞、免疫系统、Cajal间质细胞(ICC)和 所有的平滑肌肉都需要协同工作，以实现协调的运动。钙激活的氯离子通道，ANO1 在ICC中表达，是ICC电子活动所必需的。在之前的赠款周期中，有证据表明 Ano1调节ICC的增殖，Ano1的缺失与自发性胃肠动力障碍有关 肌间ICC网络中的钙瞬变通常是协调的，但会发生改变，并且高度不协调 在Ano1基因敲除小鼠中，发现了Ano1基因的启动子。Ano1如何调节钙离子瞬变和 Ano1是如何调控的尚不清楚，但已知的是，Ano1也进行 HCO3-。关于这种竞争性更新的最重要的新奇假设是，Ano1与 转运蛋白SLC4a4调节ICC钙离子瞬变，进而调节电活动，并 锌指蛋白Gli对ICC中Ano1水平的转录调控 收缩活动。这一假设将在两个特定的目标(SA)中进行检验。SA1将决定如何 肌间ICC的起搏功能受pH调节，是通过Ano1和Ano1转运碳酸氢根离子实现的 SLC4a4(NBCe1)和SA2将决定Gli对Ano1表达的转录调控如何参与 肠道起搏和胃肠动力正常。SA得到了大量初步数据的支持。1) 改变细胞外HCO3-改变细胞内钙瞬变，但不改变Ano1；2)起搏器 ICC差异表达生电Na+，HCO3-共转运蛋白SLC4a4(NBCe1)变异体c；3)遗传和 Ano1和SLC4a4的药理阻断通过HCO3-和HCO3-进入改变细胞内钙瞬变 PH的调节；4)Gli1和Gli2直接与Ano1的启动子结合；5)Ano1的表达相反 与Gli水平相关；6)Gli抑制显著增加Ano1电流；7)Gli改变上调 下游平滑肌电活动、收缩模式和胃肠道转运；以及8)单核苷酸 突变体阻止Gli与Ano1启动子结合，并与肠易激综合征易感性有关 (IBS)。为了解决总体假设，将使用各种方法，从使用 基因编码的指示分子、高速和高分辨率显微镜技术、细胞内 PH值测量，几种转基因小鼠模型，电生理学，以及 从鉴定良好的细胞中获得遗传和表观遗传测序信息的能力。其他新消息 遗传学、转录调控、表观遗传学和pH调节方面的专业知识已与 成立了一个团队，采取创新的方法来了解胃肠道起搏在健康和疾病中的作用。作为一名 先前工作的结果、初步数据和拟议的实验，在 对Ano1的调节以及Ano1如何调节钙瞬变的理解将通过 对胃肠道内外的健康和疾病的影响。

PROJECT SUMMARY/ABSTRACT The extrinsic nervous system, enteric nerves, glia, the immune system, interstitial cells of Cajal (ICC) and smooth muscle all need to work in concert to enable coordinated motility. The Ca2+ activated Cl- channel, Ano1 is expressed in ICC and is required for ICC electrical activity. In the previous grant cycles it was shown that Ano1 regulates proliferation of ICC, that loss of Ano1 is associated with GI motility disorders, that spontaneous Ca2+ transients in the myenteric ICC network are usually coordinated but are altered and highly uncoordinated in Ano1 knockout mice, and the promoter for Ano1 was identified. How Ano1 regulates Ca2+ transients and how Ano1 is regulated is not known but what is known, though underappreciated, is that Ano1 also conducts HCO3-. The overarching novel hypothesis for this competitive renewal is that Ano1, acting with the transporter Slc4a4, regulates ICC Ca2+ transients that in turn regulate electrical activity and that transcriptional regulation of Ano1 levels in ICC by the zinc finger protein Gli physiologically regulates contractile activity. The hypothesis will be tested in two specific aims (SA). SA1 will determine how pacemaker function in myenteric ICC is regulated by pH through transport of bicarbonate ions by Ano1 and Slc4a4 (NBCe1) and SA2 will determine how transcriptional regulation of Ano1 expression by Gli contributes to normal intestinal pacemaking and GI motility. The SAs are supported by extensive preliminary data. 1) Changing extracellular HCO3- alters intracellular Ca2+ transients but not when Ano1 is blocked; 2) Pacemaker ICC differentially express the electrogenic Na+,HCO3- co-transporter Slc4a4 (NBCe1) variant c; 3) Genetic and pharmacologic block of Ano1 and Slc4a4 alter intracellular Ca2+ transients through entry of HCO3- and modulation of pH; 4) Gli1 and Gli2 bind directly to the promoter of Ano1; 5) Ano1 expression is inversely correlated with Gli levels; 6) Inhibition of Gli markedly increases Ano1 currents; 7) Upregulation of Gli alters downstream smooth muscle electrical activity, contractile patterns and GI transit; and 8) A single nucleotide variant prevents Gli binding to the Ano1 promoter and is linked to susceptibility to irritable bowel syndrome (IBS). To address the overarching hypothesis a variety of methods will be used, spanning from the use of genetically encoded indicator molecules, high speed and high resolution microscopy techniques, intracellular pH measurement, several genetically modified mouse models, electrophysiology, and improvements in the ability to obtain genetic and epigenetic sequencing information from well identified cells. Additional new expertise in genetics, transcriptional regulation, epigenetics and pH regulation has been integrated with the established team to take an innovative approach to understanding GI pacemaking in health and disease. As a result of previous work, the preliminary data, and the proposed experiments, a significant advance in the understanding of the regulation of Ano1 and how Ano1 regulates Ca2+ transients will be achieved with implications in both health and disease within and outside the GI tract.