Downregulation of epithelial sodium channel (ENaC) by CFTR co-expressed inXenopus oocytes is independent of Cl- conductance

Defective regulatory interactions between the cystic fibrosis conductance r egulator (CFTR) and the epithelial sodium channel (ENaC) have been implicat ed in the elevated Na+ transport rates across cystic fibrosis airway epithe lium. It has recently been proposed that ENaC downregulation by CFTR depend s on the ability of CFTR to conduct Cl- into the cell and is negligible whe n Cl- flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (I-amil) in oocytes co- expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to -60 mV, stimulating CFTR with 1 mM IBMX reduced I-amil by up to 80%, demon strating that ENaC is inhibited when Cl- is conducted out of the cell. Decr easing the level of CFTR stimulation in a single oocyte, decreased both the degree of I-amil downregulation and the CFTR-mediated plasma membrane Cl- conductance, suggesting a direct correlation. However, I-amil downregulatio n was not affected when Cl- flux across oocyte membrane was minimized by ho lding the oocyte membrane potential near the Cl- reversal potential (67% +/ - 10% inhibition at -20 mV compared to 79% +/- 4% at -60 mV) demonstrating that I-amil downregulation was independent of the amount of current flow th rough CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed th at Ca2+ is not involved in I-amil downregulation by CFTR, although Ca2+ inj ection into the cytoplasm did inhibit I-amil. These results demonstrate tha t downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitud e of Cl- transport across the plasma membrane.