Expression and Regulation of Normal and Polymorphic Epithelial Sodium Channel by Human Lymphocytes

James K. Bubien, Bracie Watson, Masood A. Khan, Anne Lynn B Langloh, Catherine M. Fuller, Bakhram Berdiev, Albert Tousson, Dale J. Benos

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

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 α-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.

Original languageEnglish
Pages (from-to)8557-8566
Number of pages10
JournalJournal of Biological Chemistry
Volume276
Issue number11
DOIs
Publication statusPublished - Mar 16 2001
Externally publishedYes

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Epithelial Sodium Channels
Lymphocytes
Amiloride
Antibodies
Sodium Channels
Sodium
Lipid bilayers
Polymerase chain reaction
RNA-Directed DNA Polymerase
Cell membranes
Gene expression
Lipid Bilayers
Purification
Reverse Transcriptase Polymerase Chain Reaction
Proteins
Fluorescence
Therapeutics
Lipids
Immunohistochemistry
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bubien, J. K., Watson, B., Khan, M. A., Langloh, A. L. B., Fuller, C. M., Berdiev, B., ... Benos, D. J. (2001). Expression and Regulation of Normal and Polymorphic Epithelial Sodium Channel by Human Lymphocytes. Journal of Biological Chemistry, 276(11), 8557-8566. https://doi.org/10.1074/jbc.M008886200

Expression and Regulation of Normal and Polymorphic Epithelial Sodium Channel by Human Lymphocytes. / Bubien, James K.; Watson, Bracie; Khan, Masood A.; Langloh, Anne Lynn B; Fuller, Catherine M.; Berdiev, Bakhram; Tousson, Albert; Benos, Dale J.

In: Journal of Biological Chemistry, Vol. 276, No. 11, 16.03.2001, p. 8557-8566.

Research output: Contribution to journalArticle

Bubien, JK, Watson, B, Khan, MA, Langloh, ALB, Fuller, CM, Berdiev, B, Tousson, A & Benos, DJ 2001, 'Expression and Regulation of Normal and Polymorphic Epithelial Sodium Channel by Human Lymphocytes', Journal of Biological Chemistry, vol. 276, no. 11, pp. 8557-8566. https://doi.org/10.1074/jbc.M008886200
Bubien, James K. ; Watson, Bracie ; Khan, Masood A. ; Langloh, Anne Lynn B ; Fuller, Catherine M. ; Berdiev, Bakhram ; Tousson, Albert ; Benos, Dale J. / Expression and Regulation of Normal and Polymorphic Epithelial Sodium Channel by Human Lymphocytes. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 11. pp. 8557-8566.
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