Cystic fibrosis is a recessive disease caused by any of 200+ mutations in the gene for CFTR, an ATP and PKA-regulated anion channel that is primarily localized to apical plasma membranes of certain epithelial cells. Can all defects of cystic fibrosis be traced to defective membrane anion conductance, or does CFTR perform other functions? if it does, what are they? We will begin to address this issue in human airway submucosal gland cells, where Cl- secretion in response to both Ca2+ and cAMP is defective. We will use patch-clamp recordings to determine if CFTR channels are the exclusive pathway for cAMP and Ca2+-stimulated Cl- secretion in primary cultures of human submucosal gland cells, or if CFTR is regulated by Ca2+ in these cells. We will also assay activation and single-channel kinetics of endogenous and recombinant CFTR channels, and will assess the influence on kinetics of factors such as PKA, PKC, ATP, ADP, temperature, membrane voltage, and inhibitors of PDE and adenosine receptors. Kinetics will also be studied for three classes of mutated CFTR channels. We hypothesize that mutations conferring pancreatic sufficiency (e.g. R117H and G551S) will confer residual plasma membrane anion permeability, while mutations conferring pancreatic insufficiency in spite of being correctly targeted to the membrane (e.g. G551D and G1349D) will display severely reduced open probabilities. and may indeed be non- conducting under physiological conditions. (Their presence in the membrane and can be established by high levels of IBMX.) Mutations causing CFTR to be retained in the ER (e.g. deltaF508 and S549l, which also produce pancreatic insufficiency), may yield a wide variety of channel kinetics when the channels reach the membrane after expression in cells with more permissive trafficking mechanisms or grown at cooler temperatures. If CF symptoms are tightly correlated with channel function, then anion conductance is either the critical feature responsible for CFTR's pleiotropic effects, or is not dissociable from GCl- in any of the mutated proteins. We will test the hypothesis that P-glycoprotein, another member of the ATPase transport factory with many features in common with CFTR, regulates a swelling-activated Cl- channel but is not itself a Cl- channel. We also propose to test the hypothesis that CFTR is a nucleotide conducting channel, and that nucleotides, perhaps originating from CFTR, might act as autacoids to activate other Cl- channels.