COBRE: UNR: MOLECULAR IDENTIF & CHAR OF CALCIUM-ACTIVATED CHLORIDE CHANNELS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 离子通道的分子生物学研究彻底改变了我们对这些生理关键蛋白质的结构和作用机制的认识。心血管系统中阴离子通道的任何全面检查都必须包括识别和表征这些电导背后的分子组分的目标。近年来，我们在心脏Cl-通道的分子识别方面取得了很大进展。我们已经鉴定出ICl.cAMP、ICl.PKC和ICl.ATP由CFTR的剪接变体编码;我们已经鉴定出ICl.vol为ClC-3;我们有关于ICl.ir和ClC-2之间关系的初步数据。然而，编码关键心血管氯电流（ICl.Ca）的离子通道的基因仍有待确定。这个卓越的生物医学研究中心将提供解决这个问题所需的工具和协作资源。我们将检验我们的一般假设，即心肌细胞中的ICl.Ca由CLCA和/或新型雌激素基因家族的成员编码。我们建议（1）确定雌激素分子形式的表达模式以及该数据如何与天然ICl. Ca的分布相关。RT-PCR和核酸酶保护研究将确定新的剪接变体是否在心血管组织中表达。(2)确定克隆的心脏CLCA和bestrophin基因在哺乳动物细胞系中功能性表达的生物物理特性，并评估它们与天然I. ClCa的关系。(3)在组织和细胞水平上确定雌激素的免疫定位，以确定表达雌激素通道的心血管细胞类型，并研究它们之间的共定位。(4)通过消除诱导型心脏特异性基因敲除小鼠中的雌激素蛋白表达，确定雌激素蛋白在I.ClCa中的生理作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Molecular biological studies of ion channels have revolutionized our perception of the structure and mechanism of action of these physiologically critical proteins. Any comprehensive examination of anion channels in the cardiovascular system must include a goal of identification and characterization of the molecular components underlying these conductances.We have made great strides in recent years towards the goal of molecular identification of cardiac Cl- channels. We have identified ICl.cAMP, ICl.PKC and ICl.ATP as being encoded by a splice variant of CFTR; we have identified ICl.vol as ClC-3; we have preliminary data for a relationship between ICl.ir and ClC-2.¿ However, the gene(s) that code for the ion channel underlying a key cardiovascular chloride current (ICl.Ca) remains to be determined. This Center of Biomedical Research Excellence will provide the tools and the collaborative resources required to address this question. We will test our general hypothesis that ICl.Ca in cardiac myocytes is encoded by a member(s) of the CLCA and/or novel bestrophin gene family. We propose to (1) Determine the expression pattern of molecular forms of bestrophins and how that data relates to the distribution of native ICl.Ca. RT-PCR and nuclease protection studies will determine if novel splice variants are expressed in cardiovascular tissues.¿ (2) Determine the biophysical properties of cloned cardiac CLCA and bestrophin genes functionally expressed in mammalian cells lines and evaluate their relationship to native I.ClCa.¿ (3)¿ Determine the immunolocalization of bestrophins at tissue and cellular levels to determine cardiovascular cell types expressing bestrophin channels and to investigate their co-localization with each other.¿ (4) Determine the physiological role bestrophin proteins play in I.ClCa in heart by eliminating bestrophin protein expression in an inducible heart-specific knockout mouse.¿