The broad goal of this renewal application is to define the nature and the functional relevance of the interactions between the CFTR chloride channel and SNAREs, the basic elements of the membrane fusion machinery. CFTR is an epithelial chloride channel that is implicated in two major diseases: cystic fibrosis (low CFTR activity) and secretory diarrhea (excessive CFTR activity). At present we know little about what molecules control CFTR function in epithelial cells, and how these intermolecular interactions integrate CFTR activity with other epithelial cell functions. We have observed that the macroscopic currents mediated by native CFTR and by disease-associated mutants in epithelial cells are tonically inhibited by syntaxin 1A, a plasma membrane-associated t-SNARE. We have also determined that syntaxin 1A inhibits CFTR currents by binding to the amino terminal tail of this ion channel (N-tail), and that this cytoplasmic tail plays a major role in controlling CFTR channel gating. In more recent preliminary studies we have observed that SNAP-23, a SNARE that forms heterodimers with syntaxin 1A, also appears to bind to the CFTR N-tail and regulate CFTR currents. On the basis of these results we will pursue three specific aims. First, we will test the hypothesis that SNAP-23 directly binds to the CFTR N-tail and regulates macroscopic CFTR current (Aim 1). As part of this aim we will determine if SNAP-23 and syntaxin 1A form a ternary complex with the CFTR N-tail and cooperatively regulate CFTR currents. Second, we will test the hypothesis that syntaxin 1A and/or SNAP-23 inhibit the macroscopic currents mediated by wild type CFTR and disease-associated mutants by directly modulating the single channel gating properties of CFTR (Aim 2). Third, we will determine how interactions between CFTR channels and SNAREs help coordinate the processes of fluid and electrolyte transport, SNARE complex assembly and protein secretion in epithelial tissues (Aim 3). The results should provide new insights into how the functional activity of the CFTR chloride channel is modulated by intermolecular interactions, and how these interactions integrate CFTR activity with the other physiologie demands of an epithelial cell (e.g., protein secretion). Such information may lead to new strategies for manipulating CFTR function in cystic fibrosis and secretory diarrhea.
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