ABSTRACT: The calcium-activated chloride channel regulator CLCA4 has been associated with and suggested as a drug target for inflammatory diseases of the airway and gastrointestinal tract including asthma, COPD, cystic fibrosis, infectious diarrhea, Crohn's disease, ulcerative colitis, and some cancers. 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 a related family member, CLCA1, by showing that it is a self-cleaving metalloprotease that binds to and potentiates the calcium activated chloride channel TMEM16A. In our preliminary data we show that CLCA4 acts in a similar manner on another distinct TMEM16 family member, TMEM16B. Therefore, we are uniquely poised to generate significant advances in our understanding of CLCA4 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 CLCA4 activity and comprehensively validate that TMEM16B is the sole TMEM16 family member modulated by CLCA4. In Aim 2, we will develop a novel panel of monoclonal antibody reagents specific for the mature, cleaved forms of CLCA4 (N-CLCA4 and C-CLCA4) that can be used in several experimental formats, and carry out initial analysis of healthy and diseased airway and intestinal tissue and biological samples with them. The long-term goal of this project (beyond this pilot R03) is to develop a comprehensive mechanistic understanding of CLCA4 function in health and disease, which we believe is sorely underappreciated. Successful completion of this pilot will provide the knowledge and tools to investigate the functional roles of CLCA4 in specific diseases and to develop targeted therapeutics in subsequent projects.

PROJECT NARRATIVE (RELEVANCE): The calcium-activated chloride channel regulator 4 (CLCA4) has been associated with and suggested as a drug target for a variety of inflammatory diseases of the airway and gastrointestinal tract including asthma, COPD, cystic fibrosis, infectious diarrhea, Crohn's disease, ulcerative colitis, and some cancers. However, a detailed understanding of its biological function, required to target it in disease, has lagged far behind its association with these diseases. This is largely due to a lack of basic biochemical knowledge and useful antibody reagents specific to CLCA4. This pilot project will develop the tools and knowledge needed to understand the underappreciated role of CLCA4 in health and disease and provide a framework for developing targeted therapeutics.