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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。任何对心血管系统阴离子通道的全面检查都必须包括鉴定和表征这些传导的分子组成的目标。近年来，我们在实现心脏氯通道的分子鉴定的目标上取得了长足的进步。我们已经确定ICl.cAMP、ICl.PKC和ICl.ATP是由cftr的一个剪接变体编码的；我们已经确定ICl.Vol是ClC-3；我们已经有了ICl.IR和Clc-2之间关系的初步数据。然而，编码关键的心血管氯电流(ICl.Ca)离子通道的基因(S)仍有待确定。该生物医学研究卓越中心将提供解决这一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是由CLCA和/或新的Bestrophin基因家族的一个成员(S)编码的。我们建议：(1)确定Bestrophins分子形式的表达模式，以及该数据与天然ICl.Ca分布的关系。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)确定在哺乳动物细胞系中功能性表达的心脏CLCA和Bestrophin基因的生物物理性质，并评估它们与天然I.Clca的关系。(3)确定bestrophins在组织和细胞水平的免疫定位，以确定表达Bestrophin通道的心血管细胞类型，并研究它们相互之间的共定位。(4)通过在可诱导的心脏特异基因敲除小鼠中消除Bestrophin蛋白的表达，确定Bestrophin蛋白在I.ClCa中所起的生理作用。

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.¿