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−]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- Isc 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.

Increase in intracellular Cl? concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells

“…In that study, [Cl ) ] i was assessed using the Cl ) -sensitive fluorescence dye 6-methoxy-N-(3-sulphopropyl)quinolinium (SPQ). In MDCK cell monolayers, grown polarized and basolaterally permeabilized, Xie and Scha¨fer found a large change in [Cl ) ] i following cAMP-dependent stimulation, although in this case [Cl ) ] i decreased from 76 to 36 mM [34], rather than increasing, as in our study. While the principle finding of that study, that an increase in [Cl ) ] i inhibits ENaC, is in good agreement with our findings, the decrease in [Cl ) ] i elicited by cAMP-dependent stimulation is rather surprising.…”

“…Inhibition of ENaC by increased intracellular Cl ) has also been demonstrated recently in an MDCK cell clone expressing high levels of retrovirally expressed ENaC [34]. In that study, [Cl ) ] i was assessed using the Cl ) -sensitive fluorescence dye 6-methoxy-N-(3-sulphopropyl)quinolinium (SPQ).…”

CFTR structure and function: is there a role in the kidney?

“…In addition, Kunzelman and collaborators (Konig et al 2001;Kunzelmann 2003) have shown that the inhibitory effect of CFTR can be mimicked by coexpression of other anion channels (ClC-0 and ClC-2) or treatment with amphotericin B, and this effect can be explained by an increase of Cl − currents or an intracellular Cl − ([Cl − ]i). Some authors have been shown that an increase in [Cl − ]i inhibits ENaC activity in FL-MDCK cells (MDCK cells that had been transfected with rat ENaC subunits containing the FLAG epitope in their extracellular loops; Morris and Schafer 2002); however, this effect cannot explain the effect of Cl − secretion on Na + absorption with cAMP treatment because stimulation of Cl − secretion by cAMP results in a fall rather than a rise in [Cl − ]i (Xie and Schafer 2004). Moreover, in the Xenopus oocyte expression system, CFTR activation inhibits ENaC at a continuous holding potential at which CFTR mediates inward currents corresponding to Cl − efflux (Konstas et al 2003).…”

Cl– Interference with the Epithelial Na+ Channel ENaC

“…Our initial work suggested that influx of Cl Ϫ into the oocyte rather than the efflux is inhibiting ENaC (9). Although a role of Cl Ϫ for the inhibition of ENaC by CFTR was not found in all studies (19,35), subsequent work in both epithelial tissues and Xenopus oocytes confirmed that intracellular Cl Ϫ ions decrease expression of ENaC protein (36) and acutely inhibit ENaC currents (15,27,30,37). Recent studies with polarized epithelial cells expressing ENaC along with CFTR and Ca 2ϩ -activated Cl Ϫ channels, demonstrate an increase in intracellular Cl Ϫ by stimulation of CFTR or Ca 2ϩ -dependent Cl Ϫ channels (38).…”