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

Ryan G. Morris's present address is National Institutes of Health, National Heart, Lung, and Blood Institute, Laboratory of Kidney and Electrolyte Metabolism, Bethesda, MD 20892.

Portions of this work were previously published in abstract form (Morris, R.G., and J.A. Schafer. 2000. J. Am. Soc. Nephrol. 11:34A; and Morris, R.G., and J.A. Schafer. 2001. J. Am. Soc. Nephrol. 12:37A).

Abbreviations used in this paper: ADH, antidiuretic hormone; AS-I_sc, amiloride-sensitive short-circuit current; B_max, maximal specific binding; CCD, cortical collecting duct; CPT, 8-p-chlorophenylthio; DMEM, Dulbecco's modified Eagle's medium; ENaC, epithelial (amiloride-sensitive) Na⁺ channel; FLAG, octapeptide epitope DYKDDDDY; IBMX, isobutylmethylxanthine; I_sc, short-circuit current; MDCK, Madin-Darby canine kidney; PKA, protein kinase A; R_T, transepithelial resistance; V_T, transepithelial voltage.

In the course of the present experiments, we also measured whole cell amiloride-sensitive currents in Xenopus oocytes expressing wild-type and FLAG-labeled rat ENaC subunits. Using a variety of cAMP analogs, we were unable to show any stimulatory effect, even at high concentrations in the presence of IBMX. Most likely the lack of effect is due to the high rate of endogenous cAMP production that we confirmed in these oocytes (unpublished data).

Plots of specific binding as a function of antibody concentration are presented and analyzed using the Michaelis-Menten equation as described in materials and methods. This approach is used in preference to a Scatchard plot (bound/free versus free antibody concentration) because the Michaelis-Menten plot shows the actual data obtained and its variation, and because the nonlinear regression of the data according to the Michaelis-Menten relationship avoids the bias introduced into least-squares fitting by the linearized Scatchard relationship. Nevertheless, the results of Scatchard analysis were quite comparable with those obtained from nonlinear regression using the Michaelis-Menten equation. In the case of Fig. 4, linear regression of the Scatchard plot gave k_0.5 and B_max estimates of, respectively, 9.2 ± 2.2 nM and 8.0 ± 0.8 fmol/cm² (r = 0.90, P < 0.001).

Drs. J.K. Bubien and Z.-H. Zhou in our department were kind enough to conduct preliminary patch clamp studies of the cells used in these studies using methods they have described previously (Bubien et al., 2001; Zhou and Bubien, 2001). In detached patches from α_Fβ_Fγ_F MDCK cells, they observed one to three channels per patch with conductances in the range of 5–10 pS, NP_o products of 0.5–1.5, and the long open and closed intervals characteristic of ENaC channels with or without the FLAG label (Firsov et al., 1996; Garty and Palmer, 1997). In one inside-out patch from an α_Fβ_Fγ_F MDCK cell, the I-V relationship was linear in the range of ±80 mV with a slope conductance of 8.6 pS. In whole-cell patches of untreated α_Fβ_Fγ_F MDCK cells, there was a basal level of ENaC activity that was markedly increased by cAMP. In whole-cell patch clamps of parental cells, there was no evidence of ENaC-like channel activity in the presence or absence of cAMP treatment (Bubien, J.K., and A.L. Birmingham, personal communication).

Scatchard analyses of these data gave k_0.5 estimates of: 4.6 ± 0.8 (control) and 3.5 ± 0.6 (+cAMP), and B_max estimates of 1.9 ± 0.5 (control) and 2.9 ± 0.5 (+cAMP).