4.5 Article

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

Journal

AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
Volume 300, Issue 4, Pages F887-F897

Publisher

AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00648.2010

Keywords

cell surface; membrane; oocyte; self-inhibition

Funding

  1. Israel Ministry of Health
  2. Peter Simpson Family Fund

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Edelheit O, Hanukoglu I, Dascal N, Hanukoglu A. Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis. Am J Physiol Renal Physiol 300: F887-F897, 2011. First published January 5, 2011; doi:10.1152/ajprenal.00648.2010.-Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human alpha-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type beta- and gamma-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na+ conductance (assayed by the two-electrode voltage-clamp method), Na+-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to gamma-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na+ conductance (to 40-80% of WT). Mutation of only six residues showed a significant effect on the Na+ self-inhibition time constant (tau). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression (r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with tau = similar to 30% of WT, and Asp393 and Glu530 with tau = similar to 170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of alpha-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na+ self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na+ self-inhibition, most are essential for transport to the oocyte cell surface.

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