Inhibition of ENaC by intracellular Cl−in an MDCK clone with high ENaC expression

Abstract

We examined the effects of intracellular Cl−concentration ([Cl−]i) on the epithelial Na channel (ENaC) in a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) with a high rate of Na+transport produced by stable retroviral transfection with rENaC subunits (Morris RG and Schafer JA. J Gen Physiol 120: 71–85, 2002). Treatment with cAMP (100 μM 8-cpt-cAMP plus 100 μM IBMX) stimulated ENaC-mediated Na+absorption as well as Cl−secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in α-toxin-permeabilized monolayers to have the anion selectivity sequence NO3−> Br−> Cl−> I−. With the use of FL-MDCK monolayers in which the basolateral membrane was permeabilized by nystatin, the ENaC conductance of the apical membrane [determined from the amiloride-sensitive short-circuit current (AS- Isc) driven by an apical-to-basolateral Na+concentration gradient] was progressively inhibited by increasing the [Cl−] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [Cl−] in the apical solution. This inhibitory effect of [Cl−]ioccurred regardless of the presence or absence of net Cl−transport. However, from fluorometric measurements using the Cl−-sensitive dye 6-methoxy- N-(3-sulfopropyl)-quinolinium in intact FL-MDCK monolayers on permeable supports, cAMP, which activates both Na+absorption and Cl−secretion, produced a decrease of [Cl−]ifrom 76 ± 14 to 36 ± 8 mM ( P = 0.03). Thus it might be expected that activation of Cl−secretion by cAMP would lead to stimulation rather than inhibition of ENaC. In the nystatin-treated monolayers, an increase in [Cl−]ifrom 15 to 145 mM decreased AS- Iscfrom 24.5 ± 1.0 to 10.2 ± 1.6 μA/cm2. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 ± 0.16 to 1.32 ± 0.17 fmol/cm2and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 μA/fmol.