REGULATION OF EPITHELIAL NA-1 CORTICAL COLLECTING DUCT CELLS( CHANNELS FROM M)

The M-1 cell line is derived from the mouse cortical collecting duct a nd 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 m u 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 > a miloride much greater than dimethylamiloride. Under our experimental c onditions, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cycli c monophosphate (cAMP) had no detectable effects on I-sc or g(T). Incr easing apical Na+ entry with nystatin increased I-sc. The possible reg ulation 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. Perf usion 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 consist ent with the concept that PKA stimulates ENaCs by phosphorylating a si te with access to but not within the apical membrane patch during cell -attached and excised-patch studies.