Inhibition of ENaC by intracellular [Cl.sup.-] in an MDCK clone with high ENaC expression

Inhibition of ENaC by intracellular [Cl.sup.-] in an MDCK clone with high ENaC expression. Am J Physiol Renal Physiol 287:F722-F731, 2004. First published May 25, 2004:10.1152/ajprenal.00135.2004.--We examined the effects of intracellular Cl concentration ([[[Cl.sup.-]].sub.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.sup.+] 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 [micro]M 8-cpt-cAMP plus 100 [micro]M IBMX) stimulated ENaC-mediated [Na.sup.+] absorption as well as Cl secretion via cystic fibrosis transmembrane conductance regulator, which was characterized in [alpha]-toxin-permeabilized monolayers to have the anion selectivity sequence N[O.sup.-.sub.3] > Br > [Cl.sup.-] > [I.sup.-]. 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.sub.sc]) driven by an apical-to-basolateral [Na.sup.+] concentration gradient] was progressively inhibited by increasing the [[Cl.sup.-]] in the basolateral solution (and hence in the cytosol), but it was insensitive to the [[Cl.sup.-]] in the apical solution. This inhibitory effect of [[[Cl.sup.-].sub.i] occurred regardless of the presence or absence of net [Cl.sup.-] 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.sup.+] absorption and [Cl.sup.-] secretion, produced a decrease of [[[Cl.sup.-]].sub.i] from 76 [+ or -] 14 to 36 [+ or -] 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.sup.-]].sub.i] from 15 to 145 mM decreased AS-[I.sub.sc] from 24.5 [+ or -] 1.0 to 10.2 [+ or -] 1.6 [micro]A/[cm.sup.2]. This inhibition of ENaC could be attributed to nearly proportional decreases in the density of ENaC in the apical membrane from 1.91 [+ or -] 0.16 to 1.32 [+ or -] 0.17 fmol/[cm.sup.2] and in the intrinsic channel activity (the average current per ENaC subunit) from 13.3 [+ or -] 1.2 to 8.2 [+ or -] 1.4 [micro]A/fmol. epithelial sodium channel surface density; chloride channels; cystic fibrosis transmembrane conductance regulator