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

Department of Physiology and Biophysics and Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294

Submitted 15 April 2004 ; accepted in final form 20 May 2004

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 -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-I_sc) 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^–]_i occurred 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^–]_i from 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^–]_i from 15 to 145 mM decreased AS-I_sc from 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/cm² and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 ± 1.2 to 8.2 ± 1.4 µA/fmol.

epithelial sodium channel surface density; chloride channels; cystic fibrosis transmembrane conductance regulator

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