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J Biol Chem, Vol. 274, Issue 46, 32889-32896, November 12, 1999
The NH2 Terminus of the Epithelial Sodium Channel
Contains an Endocytic Motif
Michael L.
Chalfant ,
Jerod S.
Denton ,
Anne Lynn
Langloh§,
Katherine H.
Karlson ,
Johannes
Loffing¶,
Dale J.
Benos§, and
Bruce A.
Stanton
From the 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
An epithelial sodium channel (ENaC) is composed
of three homologous subunits: , , 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.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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