Cyclic nucleotide permeability of connexin mutants related to genetic disease

描述(申请人提供)：缝隙连接为细胞间信号和脉冲传导提供了一条直接的细胞间途径，在正常生理中发挥着重要作用。然而，它们的连接蛋白突变与许多遗传性疾病有关，包括神经系统疾病、耳聋、白内障和一些皮肤病。连接蛋白43(Cx43)的突变被认为与眼齿-指发育不良(Oddd)有关，后者表现为骨畸形和伴随的神经并发症。在另一个案例中，Cx40突变与房颤和其他心律失常相关。大多数与疾病相关的突变都是单一氨基酸替代，我们假设它们可能会改变受影响的连接蛋白通道的特定膜选择性属性。我们希望对野生型Cx43和Cx40的第二信使通透性进行量化，并将它们与人类与先天性心脏病和心律失常相关的Cx43和Cx40突变进行比较。环核苷酸cAMP和cGMP在正常骨骼发育过程中起重要作用，对心肌细胞的收缩和起搏起作用。在AIM1中，将测定Cx40和Cx43形成的缝隙连接通道对第二信使cAMP和cGMP的渗透选择性。报告基因SpIH将用于监测cAMP/cGMP通透性，同时通过双全细胞膜片钳监测连接电导。AIM2将确定由Cx40(A96S、M163V和G38D)和Cx43(L90V、I130T和K134E)的疾病连锁突变形成的缝隙连接通道的环核苷酸膜选择性特性。我们将量化和比较突变型连接蛋白形成的缝隙连接通道与野生型连接蛋白通道的通透性。AIM3将表征由Cx40和Cx43突变体形成的缝隙连接通道的细胞间运输、门控特性和膜选择性特性。我们将测试通道开放概率、电压和pH依赖的门控、细胞间运输和渗透性的变化，因为这些参数代表了突变可以降低细胞间通信的大小的替代方式。在这项研究中，我们将定性和定量地比较野生型和疾病相关突变型连接蛋白对环核苷酸的选择性。拟议的研究结果将为理解PERM选择性作为疾病状态(如ODD和房颤)的潜在决定因素所起的作用奠定基础。根据AIMS 1-3的结果，我们将确定哪些通道属性(Perm选择性、开放概率或运输)对突变连接蛋白的功能改变负责。 公共卫生相关性：缝隙连接在正常生理中起着重要作用，其连接蛋白突变与许多人类遗传性疾病有关。这一建议试图定性和定量地评估野生型和疾病相关突变体连接蛋白的透膜选择性，包括对已知信号分子如环核苷酸的通透性。这项拟议的研究结果将为理解PERM选择性作为疾病状态的潜在决定因素的作用奠定基础，如眼指发育不良和心房颤动。

DESCRIPTION (provided by applicant): Gap junctions provide a direct intercellular pathway for cell-to-cell signaling and impulse conduction and play an important role in normal physiology. However, mutations in their connexin proteins have been implicated in many hereditary diseases, including nervous system disorders, deafness, cataracts and some skin diseases. Mutations in connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD) which manifests with bone deformities and accompanying neurological complications. In another case, Cx40 mutations have been correlated with atrial fibrillation and other arrhythmias. Most of the disease associated mutations are single amino acid substitutions and we hypothesize that they could potentially alter the specific perm-selectivity properties of the affected connexin channel. We wish to quantify second messenger permeability for wild-type Cx43 and Cx40 and compare them to human mutations of Cx43 and Cx40 associated with ODDD and arrhythmia. The cyclic nucleotides, cAMP and cGMP are of particular interest as both are important in normal bone development and play a role in cardiac myocyte contractility and pacing. In Aim1 the perm-selectivity properties of gap junction channels formed by Cx40 and Cx43 to second messengers cAMP and cGMP will be determined. A reporter gene SpIH will be used to monitor cAMP/cGMP permeability while simultaneously monitoring junctional conductance by dual whole cell patch clamp. Aim2 will determine the cyclic nucleotide perm-selectivity properties of gap junction channels formed by disease linked mutations in Cx40 (A96S, M163V and G38D) and Cx43 (L90V, I130T and K134E). We will quantify and compare the permeability of gap junction channels formed by mutated connexins to their wild-type counterparts. Aim3 will characterize the intercellular trafficking, gating properties and perm-selectivity properties of gap junction channels formed by Cx40 and Cx43 mutants. We will test for changes in channel open probability, voltage and pH dependent gating, intercellular trafficking and permeability as these parameters represent alternative ways that mutations could diminish the magnitude of cell-to-cell communication. In this proposal, the cyclic nucleotide perm-selectivity of wild-type and disease associated mutant connexins will be compared qualitatively and quantitatively. The results of the proposed research will establish a baseline for understanding the role of perm-selectivity as a potential determinant of disease states such as ODDD and atrial fibrillation. Based on the results of Aims 1-3, we will determine which channel properties (perm-selectivity, open probability, or trafficking) are responsible for functional alterations in mutated connexins. PUBLIC HEALTH RELEVANCE: Gap junctions play an important role in normal physiology and mutations in their connexin proteins have been implicated in many human hereditary diseases. This proposal seeks to evaluate the perm-selectivity of wild-type and disease associated mutant connexins qualitatively and quantitatively, including permeability to known signaling molecules like cyclic nucleotides. The results of the proposed research will establish a baseline for understanding the role of perm-selectivity as a potential determinant of disease states such as oculodentodigital dysplasia and atrial fibrillation.