Source
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Abstract
An epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. To elucidate the function of the cytoplasmic, NH(2) terminus of rat ENaC (rENaC) subunits, a series of mutant cDNAs was constructed and the cRNAs for all three subunits were expressed in Xenopus oocytes. Amiloride-sensitive Na(+) currents (I(Na)) were measured by the two-electrode voltage clamp technique. Deletion of the cytoplasmic, NH(2) terminus of alpha (Delta2-109), beta (Delta2-49), or gamma-rENaC (Delta2-53) dramatically reduced I(Na). A series of progressive, NH(2)-terminal deletions of alpha-rENaC were constructed to identify motifs that regulate I(Na). Deletion of amino acids 2-46 had no effect on I(Na): however, deletion of amino acids 2-51, 2-55, 2-58, and 2-67 increased I(Na) by approximately 4-fold. By contrast, deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I(Na). To evaluate the mechanism whereby Delta2-67-alpha-rENaC increased I(Na), single channels were evaluated by patch clamp. The single-channel conductance and open probability of alpha,beta,gamma-rENaC and Delta2-67-alpha,beta,gamma-rENaC were similar. However, the number of active channels in the membrane increased from 6 +/- 1 channels per patch with alpha,beta,gamma-rENaC to 11 +/- 1 channels per patch with Delta2-67-alpha,beta,gamma-rENaC. Laser scanning confocal microscopy confirmed that there were more Delta2-67-alpha,beta, gamma-rENaC channels in the plasma membrane than alpha,beta, gamma-rENaC channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocytic retrieval of channels from the plasma membrane and increased the half-life of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conclude that the cytoplasmic, NH(2) terminus of alpha-, beta-, and gamma-rENaC is required for channel activity. The cytoplasmic, NH(2) terminus of alpha-rENaC contains two key motifs. One motif regulates the endocytic retrieval of the channel from the plasma membrane. The second motif is required for channel activity.