Characterization of an epithelial sodium(+) channel from the mouse cortical collecting duct

Amiloride-sensitive currents in the mouse cortical collecting duct cell line, M-1, were characterized. Amiloride, added to the apical fluid compartment at a final concentration of 25 $\mu$M, inhibited the transepithelial short-circuit current $(I\sb{SC})$ by 91.5%. The structure-affinity sequence for inhibition of $I\sb{SC}$ by analogs of amiloride was benzamil $\u3e$ amiloride $\gg$ dimethylamiloride, consistent with the presence of high-affinity amiloride-sensitive sodium channels. To identify the single-channel basis of the amiloride-sensitive transepithelial current and to provide a framework for study of the regulation of the sodium conductance, whole-cell and single-channel patch-clamp methods were employed. The current-voltage relations of the amiloride-sensitive whole-cell currents exhibited rectification consistent with Goldman rectification. The cation permeability sequence of the whole-cell amiloride-sensitive conductance was $\rm Li\sp+ \u3e Na\sp+ \gg K\sp+ =$ N-methyl- sc D-glucamine (NMDG). Single-channel currents from low-conductance (2-5 pS) Na$\sp+$-selective and amiloride-blockable channels were examined. The cation-selectivity sequence for the single channels was identical to the sequence for the whole-cell currents. The current-voltage relations could be described by the Goldman equation over the voltage range from $-100$ mV to 20 mV. The distribution of dwell-times from records containing a single channel suggested the presence of multiple kinetically-distinct open and closed states. Rundown of channel activity in excised inside-out membrane patches could be delayed by incubating the cells in hypotonic solutions prior to patch-clamping. The hypothesis that phosphorylation of the constitutive channel by protein kinase A (PKA) results in modification of unitary sodium current was examined using the excised inside-out variant of the patch-clamp technique. Perfusion of the cytosolic face of the membrane patch with solutions containing PKA and its cofactor, MgATP did not alter the unitary current, the gating kinetics, or the current-voltage relations of the Na$\sp+$ channels. Similarly, alkaline phosphatase had no effect on the unitary properties of the channels