Alternatively spliced forms of the α subunit of the epithelial sodium channel: Distinct sites for amiloride binding and channel pore

Xiao Jiang Li, Ruo Hui Xu, William B Guggino, Solomon H Snyder

Research output: Contribution to journalArticle

Abstract

The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, α, β, and γ. Sodium conductance occurs when only the α subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (αENACa and αENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for αENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that αENACa is expressed to a lesser extent than αENAC in kidney, lung, and taste tissues. αENACa differs from αENAC by a deletion in the second transmembrane domain. Despite this deletion, αENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of αENACa resembles that of αENAC, being inhibited more potently by phenamil (K(d) = 65 nM) than amiloride. Unlike αENAC, expression of αENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or L+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the α subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in αENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.

Original languageEnglish (US)
Pages (from-to)1133-1140
Number of pages8
JournalMolecular Pharmacology
Volume47
Issue number6
StatePublished - Jun 1995

Fingerprint

Epithelial Sodium Channels
Amiloride
Binding Sites
Xenopus
Reverse Transcription
Oocytes
Kidney
Polymerase Chain Reaction
Nonsense Codon
Ribonucleases
Southern Blotting
Protein Isoforms
Nucleotides
Sodium
Amino Acids
Lung
Genes
phenylamil
Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

@article{f0e5cc1043f545a9a3f488f729985e56,
title = "Alternatively spliced forms of the α subunit of the epithelial sodium channel: Distinct sites for amiloride binding and channel pore",
abstract = "The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, α, β, and γ. Sodium conductance occurs when only the α subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (αENACa and αENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for αENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that αENACa is expressed to a lesser extent than αENAC in kidney, lung, and taste tissues. αENACa differs from αENAC by a deletion in the second transmembrane domain. Despite this deletion, αENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of αENACa resembles that of αENAC, being inhibited more potently by phenamil (K(d) = 65 nM) than amiloride. Unlike αENAC, expression of αENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or L+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the α subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in αENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.",
author = "Li, {Xiao Jiang} and Xu, {Ruo Hui} and Guggino, {William B} and Snyder, {Solomon H}",
year = "1995",
month = "6",
language = "English (US)",
volume = "47",
pages = "1133--1140",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "6",

}

TY - JOUR

T1 - Alternatively spliced forms of the α subunit of the epithelial sodium channel

T2 - Distinct sites for amiloride binding and channel pore

AU - Li, Xiao Jiang

AU - Xu, Ruo Hui

AU - Guggino, William B

AU - Snyder, Solomon H

PY - 1995/6

Y1 - 1995/6

N2 - The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, α, β, and γ. Sodium conductance occurs when only the α subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (αENACa and αENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for αENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that αENACa is expressed to a lesser extent than αENAC in kidney, lung, and taste tissues. αENACa differs from αENAC by a deletion in the second transmembrane domain. Despite this deletion, αENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of αENACa resembles that of αENAC, being inhibited more potently by phenamil (K(d) = 65 nM) than amiloride. Unlike αENAC, expression of αENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or L+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the α subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in αENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.

AB - The amiloride-sensitive epithelial sodium channel (ENAC) consists of at least three subunits, α, β, and γ. Sodium conductance occurs when only the α subunit is expressed in Xenopus oocytes, but it is greatly enhanced by coexpression of all three subunits. All three subunits have two transmembrane domains. Whether the amiloride binding site exists in the extracellular portion or a transmembrane domain has not been established. Using reverse transcription-polymerase chain reaction in rat taste tissues, we have identified two alternatively spliced transcripts of ENAC (αENACa and αENACb) with deletions of nucleotides that introduce a premature stop codon and may result in proteins shortened by 199 and 216 amino acids, respectively, at the carboxyl terminus. Genomic Southern blots indicate that a single gene accounts for αENAC and the alternatively spliced variants. Reverse transcription-polymerase chain reaction and RNase protection assays demonstrate that αENACa is expressed to a lesser extent than αENAC in kidney, lung, and taste tissues. αENACa differs from αENAC by a deletion in the second transmembrane domain. Despite this deletion, αENACa expression in transfected human embryonic kidney 293 cells or CV-1 cells augments [3H]phenamil binding. The [3H]phenamil binding of αENACa resembles that of αENAC, being inhibited more potently by phenamil (K(d) = 65 nM) than amiloride. Unlike αENAC, expression of αENACa in Xenopus oocytes fails to generate amiloride-sensitive Na+ or L+ currents. These results suggest that the amiloride binding site resides on the extracellular loop of the α subunit of ENAC and not the putative second transmembrane domain, which forms a channel pore. Heterogeneity in αENAC isoforms may contribute to the complexity of multimeric structures and functional variation of ENAC.

UR - http://www.scopus.com/inward/record.url?scp=0029041610&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029041610&partnerID=8YFLogxK

M3 - Article

C2 - 7603452

AN - SCOPUS:0029041610

VL - 47

SP - 1133

EP - 1140

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 6

ER -