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

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- I _sc) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS- I _sc measured in the same experiments. Stimulation with cAMP (20 μM 8-p-chlorothio-cAMP plus 200 μM IBMX) significantly increased AS- I _sc from 11.2 ± 1.3 to 18.1 ± 1.3 μA/cm ². M2 binding (at 1.7 nM M2) increased in direct proportion to AS- I _sc from 0.62 ± 0.13 to 1.16 ± 0.18 fmol/cm ². Based on the concentration dependence of M2 binding, the quantity of Na ⁺ channels per unit of AS- I _sc 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 <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS- I _sc to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I _sc increase produced by cAMP.