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

Abstract

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 (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.2 mS/cm2. Isc was abolished by blocking the Na+ pump with ouabain. Both Isc and transepithelial conductance (gT) were inhibited by benzamil > amiloride >> dimethylamiloride. Under our experimental conditions, vasopressin, vasopressin, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) had no detectable effects on Isc or gT. Increasing apical Na+ entry with nystatin increased Isc. 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 (Po) 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 Po, 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.

Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels

“…These minor discrepancies may be the result of batch-to-batch variability in oocytes, which we have tried to account for by normalizing our data, as well as the potentially nonlinear nature of the chemiluminescence surface expression assay compared with TEV. These data do not rule out alterations in mENaC P o or unitary conductance, but subtle changes in P o can be difficult to ascertain because P o can vary widely in ENaC (32).…”

Dexamethasone enhances phospholipase D activity in M-1 cells

“…M-1 cells, which preserve functional properties typical for cortical collecting duct principal cells in vivo, reabsorb sodium and secrete potassium through a corticosteroid regulated Na + channel (Chalfant et al, 1996). Recent studies have demonstrated that dexamethasone stimulates Na + transport in M-1 cells (Nakhoul et al, 1998).…”

External Nickel Inhibits Epithelial Sodium Channel by Binding to Histidine Residues within the Extracellular Domains of α and γ Subunits and Reducing Channel Open Probability

“…1D) and the lack of change in oocyte capacitance in response to Ni 2ϩ (31). The open probability of ENaC has been shown to be highly variable in different systems (1,95 binding leads to a decrease in ENaC P o , based on our presumption that the Ni 2ϩ -binding site is located within the extracellular region outside of the conduction pore. One model (the "gate" model) places the Ni 2ϩ -binding site within a putative gate that swings into the outer vestibule of the pore during channel closure.…”