This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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