Development of tools and knowledge to facilitate the investigation of chloride channel regulator CLCA2 in rare human diseasses

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ABSTRACT: The calcium-activated chloride channel regulator CLCA2 has been associated with and suggested as a drug target for inflammatory diseases of the airway and gastrointestinal tract including the rare disease cystic fibrosis (CF). However, a detailed understanding of its biological functions has lagged far behind its association with these diseases, largely due to complete lack of biochemical knowledge and quality antibody reagents. We recently made groundbreaking advances investigating fellow family members, CLCA1 and CLCA4, by showing that they are self-cleaving metalloproteases that potentiate the calcium activated chloride channels TMEM16A and TMEM16B, respectively. In our preliminary data we show that CLCA2 can also potentiate a calcium- activated chloride channel in human cells. Therefore, we are uniquely poised to generate significant advances in our understanding of CLCA2 function in health and disease. This pilot project will develop the knowledge base and tools required to facilitate this. In Aim 1, we will investigate the mechanism of regulation of CLCA2 activity and comprehensively identify the TMEM16 family member modulated by CLCA2. In Aim 2, we will develop a novel panel of monoclonal antibody reagents specific for the mature, cleaved forms of CLCA2 (N- CLCA2 and C-CLCA2) that can be used in several experimental formats, and carry out initial analysis of healthy and diseased CF airway tissues and cell samples with them. The long-term goal of this project (beyond this pilot R03) is to develop a comprehensive mechanistic understanding of CLCA2 function in health and disease, which could provide new therapeutic routes to treat CF and other muco-obstructive diseases, as we demonstrate in our preliminary data. Successful completion of this pilot will provide the knowledge and tools to investigate the functional roles of CLCA2 in CF, with the potential to develop targeted therapeutics in subsequent projects, as well as provide insight into CLCA2 links in asthma, COPD, infectious diarrhea, Crohn’s disease, ulcerative colitis, and some cancers.