Charged residues in the M2 region of α-hENaC play a role in channel conductance

Anne Lynn B Langloh, Bakhrom Berdiev, Hong Long Ji, Kent Keyser, Bruce A. Stanton, Dale J. Benos

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

The epithelial Na+ channel (ENaC) is a low-conductance channel that is highly selective for Na+ and Li+ over K+ and impermeable to anions. The molecular basis underlying these conduction properties is not well known. Previous studies with the ENaC subunits demonstrated that the M2 region of α-ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in α-subunit human ENaC (α-hENaC) M1 and M2 domains. Whole cell and single-channel current measurements indicated that the M2 mutations E568R, E571R, and D575R significantly decreased channel conductance but did not affect Na+:K+ permeability. We observed no functional perturbations from the M1 mutation E108R. Whole cell amiloride-sensitive current recorded from oocytes injected with the M2 α-hENaC mutants along with wild-type (wt) β- and γ-hENaC was low (46-93 nA) compared with the wt channel (1-3 μA). To determine whether this reduced macroscopic current resulted from a decreased number of mutant channels at the plasma membrane, we coexpressed mutant α-hENaC subunits with green fluorescent protein-tagged β- and γ-subunits. Confocal laser scanning microscopy of oocytes demonstrated that plasma membrane localization of the mutant channels was the same as that of wt. These experiments demonstrate that acidic residues in the second transmembrane domain of α-hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

Original languageEnglish
JournalAmerican Journal of Physiology - Cell Physiology
Volume278
Issue number2 47-2
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Epithelial Sodium Channels
Cell membranes
Oocytes
Cell Membrane
Mutation
Amiloride
Protein Subunits
Electric current measurement
Green Fluorescent Proteins
Confocal Microscopy
Permeation
Anions
Permeability
Microscopic examination
Ions
Scanning
Amino Acids
Lasers
Experiments

Keywords

  • Biotinylation
  • Channel pore
  • Confocal microscopy
  • Dual-electrode voltage clamp
  • Green fluorescent protein
  • Planar lipid bilayers
  • Site-directed mutagenesis
  • Xenopus oocytes

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Langloh, A. L. B., Berdiev, B., Ji, H. L., Keyser, K., Stanton, B. A., & Benos, D. J. (2000). Charged residues in the M2 region of α-hENaC play a role in channel conductance. American Journal of Physiology - Cell Physiology, 278(2 47-2).

Charged residues in the M2 region of α-hENaC play a role in channel conductance. / Langloh, Anne Lynn B; Berdiev, Bakhrom; Ji, Hong Long; Keyser, Kent; Stanton, Bruce A.; Benos, Dale J.

In: American Journal of Physiology - Cell Physiology, Vol. 278, No. 2 47-2, 2000.

Research output: Contribution to journalArticle

Langloh, ALB, Berdiev, B, Ji, HL, Keyser, K, Stanton, BA & Benos, DJ 2000, 'Charged residues in the M2 region of α-hENaC play a role in channel conductance', American Journal of Physiology - Cell Physiology, vol. 278, no. 2 47-2.
Langloh, Anne Lynn B ; Berdiev, Bakhrom ; Ji, Hong Long ; Keyser, Kent ; Stanton, Bruce A. ; Benos, Dale J. / Charged residues in the M2 region of α-hENaC play a role in channel conductance. In: American Journal of Physiology - Cell Physiology. 2000 ; Vol. 278, No. 2 47-2.
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