The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels

Biljana Jovov, Bakhrom K. Berdiev, Catherine M. Fuller, Hong Long Ji, Dale J. Benos

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7-8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.

Original languageEnglish
Pages (from-to)4134-4140
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number6
DOIs
Publication statusPublished - Feb 8 2002
Externally publishedYes

Fingerprint

Epithelial Sodium Channels
Serine Proteases
Trypsin
Proteolysis
Sodium Channels
Protein Subunits
Autoradiography
Polyacrylamide Gel Electrophoresis
Buffers
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels. / Jovov, Biljana; Berdiev, Bakhrom K.; Fuller, Catherine M.; Ji, Hong Long; Benos, Dale J.

In: Journal of Biological Chemistry, Vol. 277, No. 6, 08.02.2002, p. 4134-4140.

Research output: Contribution to journalArticle

Jovov, Biljana ; Berdiev, Bakhrom K. ; Fuller, Catherine M. ; Ji, Hong Long ; Benos, Dale J. / The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 6. pp. 4134-4140.
@article{3bbb8baaacc548d19484984ff40a0356,
title = "The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels",
abstract = "Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7-8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.",
author = "Biljana Jovov and Berdiev, {Bakhrom K.} and Fuller, {Catherine M.} and Ji, {Hong Long} and Benos, {Dale J.}",
year = "2002",
month = "2",
day = "8",
doi = "10.1074/jbc.M108354200",
language = "English",
volume = "277",
pages = "4134--4140",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

TY - JOUR

T1 - The serine protease trypsin cleaves C termini of β- and γ-subunits of epithelial Na+ channels

AU - Jovov, Biljana

AU - Berdiev, Bakhrom K.

AU - Fuller, Catherine M.

AU - Ji, Hong Long

AU - Benos, Dale J.

PY - 2002/2/8

Y1 - 2002/2/8

N2 - Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7-8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.

AB - Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7-8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.

UR - http://www.scopus.com/inward/record.url?scp=0037040194&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037040194&partnerID=8YFLogxK

U2 - 10.1074/jbc.M108354200

DO - 10.1074/jbc.M108354200

M3 - Article

VL - 277

SP - 4134

EP - 4140

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -