Regulation of epithelial Na+ channels from M-1 cortical collecting duct cells

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na+-selective channel activity of the alpha,beta,gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (I-sc) across M-1 monolayers was 89 +/- 4 mu A/cm(2), and the transepithelial conductance was 2.1 +/- 0.2 mS/cm(2). I-sc was abolished by blocking the Na+ pump with ouabain. Both I-sc and transepithelial conductance (g(T)) were inhibited by benzamil > amiloride much greater than dimethylamiloride. Under our experimental conditions, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on I-sc or g(T). Increasing apical Na+ entry with nystatin increased I-sc. The possible regulation of the M-1 Na+ channel by cAMP-activated protein kinase A (PKA) was further examined with excised inside-out patches. The open-time probability (P-o) was not fixed, displaying substantial variance. Perfusion with ATP itself, with the catalytic subunit of PKA with ATP, or with alkaline phosphatase had no consistent effect on P-o, the unitary current, or the kinetics of the M-1 Na+ channel. The data are consistent with the concept that PKA stimulates ENaCs by phosphorylating a site with access to but not within the apical membrane patch during cell-attached and excised-patch studies.