Epithelial Sodium Channel Pore Region

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 sulfhydrylreactive methanethiosulfonate derivatives ( (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 ␣-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.Epithelial sodium channels (ENaCs) 1 are composed of three homologous subunits, termed ␣-, ␤-, and ␥ENaC (1, 2). These subunits assemble to form a hetero-oligomeric, Na ϩ -selective ion channel with a subunit stoichiometry of 2␣:1␤:1␥ (3, 4), although an alternative subunit stoichiometry has been proposed (5, 6). All three Na ϩ channel subunits have cytoplasmic amino and carboxyl termini, two transmembrane domains (termed M1 and M2), and a large ectodomain (7-9). Previous studies have shown that selected point mutations within the pore region preceding M2 of each subunit altered functional properties of the channel, including cation selectivity, single channel conductance, and sensitivity to the blocker amiloride (4, 10 -14). Specific mutations of residues in a conserved threeresidue tract, (G/S)XS (where X is Ser, Gly, or Cys), within the pore region of the three ENaC subunits, rendered channels K ϩ -permeable. Snyder et al. (15) examined the accessibility of a sulfhydryl-reactive methane thiosulfonate (MTS) derivative to substituted cysteine residues within the pore region of human ␥ENaC, and they proposed a structural model of the channel pore similar to that proposed by Kellenberger et al. (11) but distinct from the resolved structure of the KcsA K ϩ channel pore (16).We previously reported that selected cysteine substitutions within the carboxyl-terminal domain of the pore region of mouse ␣ENaC (␣Ser 580 -␣Ser 592 ) altered the cation selectivity and amiloride sensitivity of the channel and proposed that this region forms the selectivity filter of the channel (14). In the current study, we systematically examined accessibility of sulfhydryl reagents to ␣␤␥mENaCs with engineered cysteine within the 24-residue pore region of the ␣-subunit. 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 Na ϩ currents. In contrast, we observed a significant increase in amiloride-sensitive Na ϩ currents following the external application of MTS derivatives or Cd 2ϩ when cysteine residues were introduced at selected sites within the amino-terminal portion of the pore region of ␣mENaC. The pattern of distribution of cysteine mutations that led to MTS-induced activation of Na ϩ currents suggests that this region has an ␣-helical structure. In addition, the activation of ␣S580C␤␥ by an MTS reage...

Arachidonic Acid Regulates Surface Expression of Epithelial Sodium Channels

“…Immediately following the washout of MTSET, whole cell currents increased in both control and in ETYAtreated oocytes, reaching a plateau after 4 min. These data suggested that MTSET produced a non-covalent inhibition of ␣S580C␤␥ currents that was reversed after washout, in agreement with previous observations (19), in addition to activation of channels by covalent modification (20). The subsequent rate of decrease of benzamil-sensitive whole cell Na ϩ current, representing internalization of channels, was significantly greater in oocytes treated with ETYA (Ϫ2.3 ϫ 10 Ϫ2 Ϯ 0.2 ϫ 10 Ϫ2 min Ϫ1 , R ϭ 0.87, n ϭ 6) when compared with controls (Ϫ0.3 ϫ …”

“…Covalent modification of this mutant by MTSET resulted in a channel open probability that approached 1 (20), and subsequent decreases in whole cell Na ϩ currents should reflect channel internalization. Oocytes expressing ␣S580C␤␥ were perfused for 4 min in TEV solution, and subsequently for 4 min with a TEV solution containing 1 mM MTSET to modify accessible cysteine residues.…”

“…Rate of ENaC Internalization Is Increased by ETYA-A recent study from our group (20) demonstrated that MTSET treatment of oocytes expressing ␣S580C␤␥ led to a change from a low open probability state to a channel open probability approaching 1.0 in conjunction with a reduction in single channel conductance. The time-dependent loss of benzamil-sensitive whole cell Na ϩ currents in oocytes expressing ␣S580C␤␥ and treated with MTSET reflects both the internalization of channels with a high open probability (lower conductance), as well as delivery of unmodified (low open probability, higher conductance) channels to the cell surface, and was used to examine ENaC endocytosis.…”

Extracellular Zn2+ Activates Epithelial Na+ Channels by Eliminating Na+ Self-inhibition

“…External Zn 2ϩ Has No Effect on ENaCs with a High Open Probability-We previously reported that ␣S580C␤␥ channels following modification by external MTSET have a high open probability and do not exhibit Na ϩ self-inhibition (9,19). If the stimulatory effect of external Zn 2ϩ on ENaC currents is due to an increase in channel open probability that would occur with a loss of Na ϩ self-inhibition, the effect of Zn 2ϩ on ENaC currents should be abolished in oocytes expressing ␣S580C␤␥ following MTSET treatment.…”

“…9, B and C). In both models, Na ϩ binds to a site within the extracellular allosteric regulatory sites (EARS) of the ENaC ECLs that we previously proposed (9) and induces local conformational changes that are transmitted to a putative gate located at the outer pore of the channel (19,24,25). Working model 1 was generated to emphasize an overlapping binding site for Na ϩ and Zn 2ϩ within the EARS.…”