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J. Biol. Chem., Vol. 276, Issue 11, 8557-8566, March 16, 2001
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From the Departments of Physiology and Biophysics, Cell Biology,
and Gerentology and Geriatric Medicine, University of Alabama,
Birmingham, Alabama 35294
Gene expression, protein expression, and function
of amiloride-sensitive sodium channels were examined in human
lymphocytes from normal individuals and individuals with Liddle's
disease. Using reverse transcriptase polymerase chain reactions,
expression of all three cloned epithelial sodium channel (ENaC)
subunits was detected in lymphocytes. Polyclonal antibodies to bovine
Expression and Regulation of Normal and Polymorphic
Epithelial Sodium Channel by Human Lymphocytes*
,
-ENaC bound to the plasma membrane of normal and Liddle's
lymphocytes. A quantitative analysis of fluorescence-tagged ENaC
antibodies indicated a 2.5-fold greater surface binding of the
antibodies to Liddle's lymphocytes compared with normal lymphocytes.
The relative binding intensity increased significantly (25%;
p < 0.001) for both normal and Liddle's cells after
treatment with 40 µM 8-CPT-cAMP. Amiloride-sensitive
whole cell currents were recorded under basal and cAMP-treated
conditions for both cell types. Liddle's cells had a 4.5-fold larger
inward sodium conductance compared with normal cells. A specific 25%
increase in the inward sodium current was observed in normal cells in
response to cAMP treatment. Outside-out patches from both cell types
under both treatment conditions revealed no obvious differences in the
single channel conductance. The Popen was
4.2 ± 3.9% for patches from non-Liddle's cells, and
27.7 ± 5.4% in patches from Liddle's lymphocytes.
Biochemical purification of a protein complex, using the same
antibodies used for the immunohistochemistry, yielded a functional
sodium channel complex that was inhibited by amiloride when
reconstituted into lipid vesicles and incorporated into planar lipid
bilayers. These four independent methodologies yielded findings
consistent with the hypotheses that human lymphocytes express
functional, regulatable ENaC and that the mutation responsible for
Liddle's disease induces excessive channel expression.
*
This work was supported by National Institutes of Health
Grants DK 37207 (to D. J. B.) and DK52789 (to J. K. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed. Tel.: 205-934-6214; E-mail: bubien@uab.edu.
§
Present address: Dept. of Biol. Chem., Johns Hopkins School of
Medicine, Baltimore, MD 21205.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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