Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is vital for Cl(-) and HCO(3)(-) transport in many epithelia. As the HCO(3)(-) concentration in epithelial secretions varies and can reach as high as 140 mm, the lumen-facing domains of CFTR are exposed to large reciprocal variations in Cl(-) and HCO(3)(-) levels. We have investigated whether changes in the extracellular anionic environment affects the activity of CFTR using the patch clamp technique. In fast whole cell current recordings, the replacement of 100 mm external Cl(-) ((Cl(o)(-))) with HCO(3)(-), Br(-), NO(3)(-), or aspartate(-) inhibited inward CFTR current (Cl(-) efflux) by approximately 50% in a reversible manner. Lowering Cl(o)(-) alone by iso-osmotic replacement with mannitol also reduced Cl(-) efflux to a similar extent. The maximal inhibition of CFTR current was approximately 70%. Raising cytosolic calcium shifted the Cl(-) dose-inhibition curve to the left but did not alter the maximal current inhibition observed. In contrast, a reduction in the internal [Cl(-)] neither inhibited CFTR nor altered the block caused by reduced Cl(o)(-). Single channel recordings from outside-out patches showed that lowering Cl(o)(-) markedly reduced channel open probability with little effect on unitary conductance. Together, these results indicate that alterations in Cl(o)(-) alone and not the Cl(-)/HCO(3)(-) ratio regulate the gating of CFTR. Physiologically, our data have implications for current models of epithelial HCO(3)(-) secretion and for the control of pH at epithelial cell surfaces.
Highlights
Cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cyclic AMP activated epithelial ClϪ channel, the mutation of which causes the potentially fatal inherited disease cystic fibrosis (CF) [1]
External HCO3Ϫ Inhibits Human CFTR Currents—Results presented in Fig. 1 demonstrate that extracellular HCO3Ϫ inhibits human CFTR expressed in Chinese hamster ovary (CHO) or baby hamster kidney (BHK) cells in a manner similar to that observed for CFTR in native guinea pig pancreatic duct cells [26]
The external face of CFTR is exposed to wide variations in luminal pH, [HCO3Ϫ], and [ClϪ] that occur normally at the epithelial cell surface
Summary
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP activated epithelial ClϪ channel, the mutation of which causes the potentially fatal inherited disease cystic fibrosis (CF) [1]. These authors found that reducing CloϪ to below 20 mM caused a remarkable time-dependent increase in the OHϪ/HCO3Ϫ permeability of oocytes transfected with CFTR This switch in OHϪ/HCO3Ϫ selectivity was suggested to arise from a conformational change in the CFTR protein, which may involve an external ClϪ binding site on the ion channel [27]. This effect of CloϪ is consistent with our previous proposal [26] that an extracellular “anion” binding site on CFTR is important for modulating channel function
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