Abstract
Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.
Highlights
From the Departments of Medicine, Physiology and §Pathology, School of Medicine, University of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104
Secondary structure predictions of the pore region suggest that the aminoterminal portion may exist as either ␣-helix or -sheet, whereas the carboxyl-terminal portion appears to be more irregular in structure
Wild type ␣␥mENaC responded to 2.5 mM MTSEA with a partial inhibition of whole cell Naϩ currents (I/I0 ϭ 0.75 Ϯ 0.05, n ϭ 17; Fig. 2A)
Summary
We previously reported that selected cysteine substitutions within the carboxyl-terminal domain of the pore region of mouse ␣ENaC (␣Ser580–␣Ser592) altered the cation selectivity and amiloride sensitivity of the channel and proposed that this region forms the selectivity filter of the channel [14]. Channels with selected cysteine mutations within the carboxyl-terminal portion of the pore region responded to the external application of MTS derivatives with an inhibition of amiloride-sensitive Naϩ currents. We observed a significant increase in amiloride-sensitive Naϩ currents following the external application of MTS derivatives or Cd2ϩ when cysteine residues were introduced at selected sites within the amino-terminal portion of the pore region of ␣mENaC. Our results suggest that the pore region has a role in ENaC gating
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