Am J Physiol Cell Physiol AJP: Heart and Circulatory Physiology
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Am J Physiol Cell Physiol 278: C277-C291, 2000;
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Vol. 278, Issue 2, C277-C291, February 2000

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

Anne Lynn B. Langloh1, Bakhrom Berdiev1, Hong-Long Ji1, Kent Keyser2, Bruce A. Stanton3, and Dale J. Benos1

Departments of 1 Physiology and Biophysics and 2 Physiological Optics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005; and 3 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755

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 alpha -ENaC is critical to channel function. Here we examine the effects of reversing the negative charges of highly conserved amino acids in alpha -subunit human ENaC (alpha -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 alpha -hENaC mutants along with wild-type (wt) beta - and gamma -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 alpha -hENaC subunits with green fluorescent protein-tagged beta - and gamma -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 alpha -hENaC affect ion permeation and are thus critical components of the conductive pore of ENaC.

site-directed mutagenesis; Xenopus oocytes; dual-electrode voltage clamp; planar lipid bilayers; green fluorescent protein; biotinylation; confocal microscopy; channel pore


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