cAMP Increases Density of ENaC Subunits in the Apical Membrane of MDCK Cells in Direct Proportion to Amiloride-sensitive Na+ Transport

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Abstract
Antidiuretic hormone and/or cAMP increase Na+ transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na+ transport could be explained quantitatively by an increased density of ENaC Na+ channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat α-, β-, and γENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. Proc. Natl. Acad. Sci. USA. 93:15370–15375). The density of ENaC subunits was quantified by specific binding of 125I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4–8 nM. Transepithelial Na+ transport was measured as the amiloride-sensitive short-circuit current (AS-Isc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-Isc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS-Isc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm2. M2 binding (at 1.7 nM M2) increased in direct proportion to AS-Isc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm2. Based on the concentration dependence of M2 binding, the quantity of Na+ channels per unit of AS-Isc was calculated to be the same in the presence and absence of cAMP, 0.23 ± 0.04 and 0.21 ±0.05 fmol/μA, respectively. These values would be consistent with a single channel conductance of ∼5 pS (typically reported for ENaC channels) only if the open probability is Isc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the Isc increase produced by cAMP.