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J Biol Chem, Vol. 274, Issue 46, 32889-32896, November 12, 1999
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From the An epithelial sodium channel (ENaC) is composed
of three homologous subunits: Department of Physiology, Dartmouth Medical
School, Hanover, New Hampshire 03755, the § Department
of Physiology and Biophysics, University of Alabama, Birmingham,
Alabama 35233, and the ¶ Institute of Anatomy, University of
Zurich, Zurich, Switzerland CH-8057
, 
, and 
. To elucidate the
function of the cytoplasmic, NH2 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 (INa)
were measured by the two-electrode voltage clamp technique. Deletion of
the cytoplasmic, NH2 terminus of (
2-109), (2-49), or -rENaC (2-53) dramatically reduced
INa. A series of progressive, NH2-terminal
deletions of -rENaC were constructed to identify motifs that
regulate INa. Deletion of amino acids 2-46 had no effect
on INa: however, deletion of amino acids 2-51, 2-55,
2-58, and 2-67 increased INa by ~4-fold. By contrast,
deletion of amino acids 2-79, 2-89, 2-100, and 2-109 eliminated
INa. To evaluate the mechanism whereby 2-67--rENaC
increased INa, single channels were evaluated by patch
clamp. The single-channel conductance and open probability of
,,-rENaC and 2-67-,,-rENaC were similar. However,
the number of active channels in the membrane increased from 6 ± 1 channels per patch with ,,-rENaC to 11 ± 1 channels
per patch with 2-67-,,-rENaC. Laser scanning confocal
microscopy confirmed that there were more 2-67-,,-rENaC channels in the plasma membrane than ,,-rENaC channels.
Deletion of amino acids 2-67 in -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,
NH2 terminus of -, -, and -rENaC is required for
channel activity. The cytoplasmic, NH2 terminus of
-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.
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