title: 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 alpha-, beta-, and gammaENaC 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 (125)I-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 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/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/microA, respectively. These values would be consistent with a single channel conductance of approximately 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.
authors.affiliation: Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Morris
authors.forename: James A
authors.initials: JA
authors.lastname: Schafer
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D004285:Dogs; D004553:Electric Conductivity; D053503:Epithelial Sodium Channels; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism / Q000502:physiology; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism* / Q000502:physiology
chemical_list: D053503:Epithelial Sodium Channels; D009842:Oligopeptides; D010455:Peptides; D020033:Protein Isoforms; D015222:Sodium Channels; D000584:Amiloride; C085706:FLAG peptide; D012964:Sodium; D000242:Cyclic AMP