Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride

C. M. Fuller, B. K. Berdiev, V. G. Shlyonsky, I. I. Ismailov, D. J. Benos

Research output: Contribution to journalArticle

Abstract

We have generated two site-directed mutants. K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loup, immediately prior to the second membrane spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channel exhibits either reduced or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ permeability, (from approximately 10:1 for wild-type αbENaC to 2:1 and 3.1 for K504E and K515E αbENaC, respectively), and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K1) for amiloride, (K) values were 0.11±0.03 μM (n = 12). 0.54±0.10 μM (n = 6), and 0.95±0.18μM (n = 7), for wild-type, K515E, and K504E αbENaC, respectively). Both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild-type αbENaC. Single channel conductance was not affected by either mutation. These observations identify a lysine-rich region between amino acid residues 495-516 of αbENaC as important to the regulation of fundamental channel properties.

Original languageEnglish
JournalFASEB Journal
Volume11
Issue number3
Publication statusPublished - 1997
Externally publishedYes

Fingerprint

Ion Channel Gating
Amiloride
ion channels
point mutation
Ion Channels
Point Mutation
mutation
mutants
lipid bilayers
Xenopus
Epithelial Sodium Channels
Membranes
cations
lysine
permeability
Mutation
Lipid bilayers
oocytes
proteins
Lipid Bilayers

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Fuller, C. M., Berdiev, B. K., Shlyonsky, V. G., Ismailov, I. I., & Benos, D. J. (1997). Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride. FASEB Journal, 11(3).

Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride. / Fuller, C. M.; Berdiev, B. K.; Shlyonsky, V. G.; Ismailov, I. I.; Benos, D. J.

In: FASEB Journal, Vol. 11, No. 3, 1997.

Research output: Contribution to journalArticle

Fuller, CM, Berdiev, BK, Shlyonsky, VG, Ismailov, II & Benos, DJ 1997, 'Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride', FASEB Journal, vol. 11, no. 3.
Fuller CM, Berdiev BK, Shlyonsky VG, Ismailov II, Benos DJ. Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride. FASEB Journal. 1997;11(3).
Fuller, C. M. ; Berdiev, B. K. ; Shlyonsky, V. G. ; Ismailov, I. I. ; Benos, D. J. / Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride. In: FASEB Journal. 1997 ; Vol. 11, No. 3.
@article{17454a319bd5414db53720ad46c14496,
title = "Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride",
abstract = "We have generated two site-directed mutants. K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loup, immediately prior to the second membrane spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channel exhibits either reduced or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ permeability, (from approximately 10:1 for wild-type αbENaC to 2:1 and 3.1 for K504E and K515E αbENaC, respectively), and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K1) for amiloride, (K) values were 0.11±0.03 μM (n = 12). 0.54±0.10 μM (n = 6), and 0.95±0.18μM (n = 7), for wild-type, K515E, and K504E αbENaC, respectively). Both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild-type αbENaC. Single channel conductance was not affected by either mutation. These observations identify a lysine-rich region between amino acid residues 495-516 of αbENaC as important to the regulation of fundamental channel properties.",
author = "Fuller, {C. M.} and Berdiev, {B. K.} and Shlyonsky, {V. G.} and Ismailov, {I. I.} and Benos, {D. J.}",
year = "1997",
language = "English",
volume = "11",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "3",

}

TY - JOUR

T1 - Point mutations in αbENaC regulate channel gating, ion conductance, and sensitivity to amiloride

AU - Fuller, C. M.

AU - Berdiev, B. K.

AU - Shlyonsky, V. G.

AU - Ismailov, I. I.

AU - Benos, D. J.

PY - 1997

Y1 - 1997

N2 - We have generated two site-directed mutants. K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loup, immediately prior to the second membrane spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channel exhibits either reduced or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ permeability, (from approximately 10:1 for wild-type αbENaC to 2:1 and 3.1 for K504E and K515E αbENaC, respectively), and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K1) for amiloride, (K) values were 0.11±0.03 μM (n = 12). 0.54±0.10 μM (n = 6), and 0.95±0.18μM (n = 7), for wild-type, K515E, and K504E αbENaC, respectively). Both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild-type αbENaC. Single channel conductance was not affected by either mutation. These observations identify a lysine-rich region between amino acid residues 495-516 of αbENaC as important to the regulation of fundamental channel properties.

AB - We have generated two site-directed mutants. K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loup, immediately prior to the second membrane spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channel exhibits either reduced or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ permeability, (from approximately 10:1 for wild-type αbENaC to 2:1 and 3.1 for K504E and K515E αbENaC, respectively), and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K1) for amiloride, (K) values were 0.11±0.03 μM (n = 12). 0.54±0.10 μM (n = 6), and 0.95±0.18μM (n = 7), for wild-type, K515E, and K504E αbENaC, respectively). Both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild-type αbENaC. Single channel conductance was not affected by either mutation. These observations identify a lysine-rich region between amino acid residues 495-516 of αbENaC as important to the regulation of fundamental channel properties.

UR - http://www.scopus.com/inward/record.url?scp=33750140488&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33750140488&partnerID=8YFLogxK

M3 - Article

VL - 11

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 3

ER -