REGULATION OF CFTR CL CHANNELS BY THE NUCLEOTIDE BINDING DOMAINS

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane C1- channel predicted to consist of five domains: two membrane spanning domains, that contribute to the formation of the channel pore; an R domain, that regulates the channel in response to phosphorylation; and two nucleotide binding domains (NBDs). The function of the NBDs in CFTR has been puzzling because they are a feature not found in other ion channels; instead, they are found in proteins involved in active transport. Moreover, many of the mutations that cause CF are found within the NBDs. We recently found that MgATP is required to open the CFTR C1 channel. In our preliminary results we found that mgatp interacts with both NBDs in CFTR, although the two NBDs are not functionally equivalent. The specific aims of this project are: 1) To learn how MgATP controls the opening and closing of the CFTR C1- channel. To fully understand how mgatp and the NBDs regulate the CFTR C1- channel, we need to know how MgATP controls channel gating. We will use the patch-clamp technique to study excised membrane patches from cells expressing recombinant wild-type CFTR and CFTR containing site-directed mutations. The results will provide an understanding, at the molecular level, of how MgATP interacts with the two NBDs to regulate CFTR. 2) We will examine the effect of compounds other than MgATP on the regulation of CFTR. These will include nonhydrolyzable analogs of ATP, nonnucleotide hydrolyzable compounds, and the products of ATP hydrolysis. The results of these studies should teach us how the NBDs interact with MgATP to maintain channel activity. 3) We will ask how CF-associated mutations in the NBDs alter C1- channel regulation. If we are to understand the pathogenesis of CF and develop innovative new therapies, we need to understand how mutations in CFTR produce defective channels. The knowledge obtained from these studies will allow a precise under- standing of such defects.