Cyclic nucleotide permeability of connexin mutants related to genetic disease

描述（由申请人提供）：间隙连接为细胞间信号传导和脉冲传导提供了直接的细胞间通路，在正常生理中发挥重要作用。然而，它们连接蛋白的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）的突变与眼齿指发育不良（ODDD）有关，ODDD表现为骨畸形和伴随的神经系统并发症。在另一个案例中，Cx40突变与心房颤动和其他心律失常有关。大多数疾病相关突变是单氨基酸取代，我们假设它们可能潜在地改变受影响连接蛋白通道的特定热选择性特性。我们希望量化野生型Cx43和Cx40的第二信使通透性，并将其与人类与ODDD和心律失常相关的Cx43和Cx40突变进行比较。环核苷酸、cAMP和cGMP尤其令人感兴趣，因为它们在正常骨骼发育中都很重要，并在心肌细胞收缩性和起搏中发挥作用。在Aim1中，Cx40和Cx43形成的间隙连接通道对第二信使cAMP和cGMP的选择性特性将被确定。报告基因SpIH将用于监测cAMP/cGMP的通透性，同时通过双全细胞膜片钳监测连接电导。Aim2将确定Cx40 （A96S、M163V和G38D）和Cx43 （L90V、I130T和K134E）中疾病相关突变形成的间隙连接通道的环核苷酸选择性特性。我们将量化并比较突变连接蛋白与野生型连接蛋白形成的间隙连接通道的渗透率。Aim3将表征Cx40和Cx43突变体形成的间隙连接通道的细胞间运输、门控特性和热选择性特性。我们将测试通道打开概率、电压和pH依赖性门控、细胞间运输和渗透性的变化，因为这些参数代表了突变可能降低细胞间通信幅度的替代方式。在本提案中，将对野生型和疾病相关突变连接蛋白的环核苷酸选择性进行定性和定量的比较。拟议的研究结果将为理解perm选择性作为疾病状态（如ODDD和房颤）的潜在决定因素的作用建立基线。基于目标1-3的结果，我们将确定哪些通道属性（永久选择性、开放概率或运输）导致突变连接蛋白的功能改变。

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.