An epithelial sodium channel (ENaC) is composed of three homologous subunit
s: 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 const
ructed and the cRNAs for all three subunits were expressed in Xenopus oocyt
es. Amiloride-sensitive Na+ currents (I-Na) were measured by the two-electr
ode voltage clamp technique. Deletion of the cytoplasmic, NH2 terminus of a
lpha (Delta 2-109), beta (Delta 2-49), or gamma-rENaC (Delta 2-53) dramatic
ally reduced I-Na. A series of progressive, NH2 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 similar to 4-fold. By contrast,
deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated I-Na. To ev
aluate the mechanism whereby Delta 2-67-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 Delta 2-67-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 Delta 2-67-alpha,beta,gamma-rENaC. Laser sca
nning confocal microscopy confirmed that there were more Delta 2-67-alpha,b
eta,gamma-rENaC channels in the plasma membrane than alpha,beta,gamma-rENaC
channels. Deletion of amino acids 2-67 in alpha-rENaC reduced the endocyti
c retrieval of channels from the plasma membrane and increased the half-lif
e of the channel in the membrane from 1.1 +/- 0.2 to 3.5 +/- 1.1 h. We conc
lude that the cytoplasmic, NH2 terminus of alpha-, beta-, and gamma-rENaC i
s required for channel activity, The cytoplasmic, NH2 terminus of alpha-rEN
aC contains two key motifs. One motif regulates the endocytic retrieval of
the channel from the plasma membrane. The second motif is required for chan
nel activity.