Triple-barrel organization of ENaC, a cloned epithelial Na+ channel

Iskander I. Ismailov, Mouhamed S. Awayda, Bakhram K. Berdiev, James K. Bubien, Joseph E. Lucas, Catherine M. Fuller, Dale J. Benos

Research output: Contribution to journalArticle

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Abstract

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the α subunit alone and those formed by α, β, and γ subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either α-rENaC alone or α- in combination with βand γ-rENaC, produced highly Na+- selective (P(Na)/P(K) = 10), amiloride-sensitive (K(i)/(umil) = 170 nM), and mechanosensitive cation channels in planar bilayers. α-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13- picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. α-, β-, γ-rENaC showed primarily the 13-pS level. α-rENaC and α,β,γ-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with >2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of α- , β-, and γ-rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.

Original languageEnglish
Pages (from-to)807-816
Number of pages10
JournalJournal of Biological Chemistry
Volume271
Issue number2
DOIs
Publication statusPublished - Jan 12 1996
Externally publishedYes

Fingerprint

Epithelial Sodium Channels
Rats
Lipid bilayers
Amiloride
Dithiothreitol
Reducing Agents
Distillation columns
Clamping devices
Lipid Bilayers
Cell membranes
Xenopus
Oocytes
Purification
Cations
Salts
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ismailov, I. I., Awayda, M. S., Berdiev, B. K., Bubien, J. K., Lucas, J. E., Fuller, C. M., & Benos, D. J. (1996). Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. Journal of Biological Chemistry, 271(2), 807-816. https://doi.org/10.1074/jbc.271.2.807

Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. / Ismailov, Iskander I.; Awayda, Mouhamed S.; Berdiev, Bakhram K.; Bubien, James K.; Lucas, Joseph E.; Fuller, Catherine M.; Benos, Dale J.

In: Journal of Biological Chemistry, Vol. 271, No. 2, 12.01.1996, p. 807-816.

Research output: Contribution to journalArticle

Ismailov, II, Awayda, MS, Berdiev, BK, Bubien, JK, Lucas, JE, Fuller, CM & Benos, DJ 1996, 'Triple-barrel organization of ENaC, a cloned epithelial Na+ channel', Journal of Biological Chemistry, vol. 271, no. 2, pp. 807-816. https://doi.org/10.1074/jbc.271.2.807
Ismailov II, Awayda MS, Berdiev BK, Bubien JK, Lucas JE, Fuller CM et al. Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. Journal of Biological Chemistry. 1996 Jan 12;271(2):807-816. https://doi.org/10.1074/jbc.271.2.807
Ismailov, Iskander I. ; Awayda, Mouhamed S. ; Berdiev, Bakhram K. ; Bubien, James K. ; Lucas, Joseph E. ; Fuller, Catherine M. ; Benos, Dale J. / Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 2. pp. 807-816.
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