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

Address correspondence to James A. Schafer, Department of Physiology and Biophysics, 1918 University Boulevard, Room 958 MCLM, Birmingham, AL 35294-0005. Fax: (205) 934-5787; E-mail: jschafer{at}uab.edu

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 -, ß-, and ENaC 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/cm². 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/cm². 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.

Key Words: retroviral transfection • FLAG epitope • channel number • membrane trafficking • short-circuit current

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