On the Molecular Basis of Ion Permeation in the Epithelial Na

The epithelial Na 1 channel (ENaC) is highly selective for Na 1 and Li 1 over K 1 and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two a , one b , and one g homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of b and g subunits, which precedes the second putative transmembrane a helix (M2). A residue in the a subunit ( a S589) at the NH 2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li 1 than Na 1 ions. The concentration of half-maximal unitary conductance is 38 mM for Na 1 and 118 mM for Li 1 , a kinetic property that can account for the differences in Li 1 and Na 1 permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of a , b , and g subunits ( a G587, b G529, g S541) decreases the Li 1 /Na 1 selectivity by changing the apparent channel affinity for Li 1 and Na 1 . Fitting single-channel data of the Li 1 permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li 1 from an outer ion binding site through the selectivity filter. Mutation of b G529 to Ser, Cys, or Asp made ENaC partially permeable to K 1 and larger ions, similar to the previously reported a S589 mutations. We conclude that the residues a G587 to a S589 and homologous residues in the b and g subunits form the selectivity filter, which tightly accommodates Na 1 and Li 1 ions and excludes larger ions like K 1 . key words: epithelial Na 1 channel • Xenopus oocyte • pore • selectivity • ion channel i n t r o d u c t i o n The highly selective epithelial sodium channel (ENaC) 1 in the apical membrane of epithelial cells represents the predominant pathway in mediating sodium reabsorption in the distal nephron, the colon, and the lung (Garty and Palmer, 1997). This electrogenic vectorial transport of Na 1 is accomplished by a two-step transport system involving the apical ENaC and the basolateral Na 1 -K 1 pump. In the distal nephron, ENaC activity is regulated by aldosterone and vasopressin, serving to maintain Na 1 balance, extracellular volume, and blood pressure. The functional characteristics of ENaC have been studied in isolated renal tubular segments and in recombinant expression systems using patch-clamp techniques. ENaC is a small 4–6-pS conductance channel in isotonic NaCl with high selectivity for Na 1 and Li 1 over K 1 (permeability ratios P Li / P Na . 1 and P Na / P K . 100) and slow gating kinetics. ENaC currents are blocked by submicromolar concentrations of amiloride. ENaC belongs to a new class of channel proteins called the ENaC/DEG superfamily, which includes a variety of proteins involved in mechanotransduction and neurotransmission, and is found in nematodes, flies, snails, and mammals (for review see Tavernarakis and Driscoll, 1997; Waldmann and Lazdunski, 1998). Subunits of this superfamily coassemble usually within subfamilies into Na 1 -preferring or -selective multimeric channels that are either constitutively active (e.g., ENaC), activated by mechanical stimuli (as postulated for Caenorhabditis elegans degenerins), activated by a peptide (FMRFamide peptide–gated Na 1 channel, FANaC; Lingueglia et al., 1995), or activated by protons (ASIC). ENaC is a heterotetramer, and made of two a , one b , and one g homologous subunits arranged around the channel pore in an abag configuration (Canessa et al., 1994b; Firsov et al., 1998). Each homologous subunit has two transmembrane spanning segments (M1 and M2) with intracellular NH 2 and COOH termini leaving a large extracellular hydrophilic loop, as illustrated in Fig. 1 A (Canessa et al., 1994a; Renard et al., 1994). Based on sequence comparisons, current models predict that the second transmembrane spanning segment of ENaC forms an a helix starting with a conserved Ser residue ( a S589) and extending 22 residues further downstream in the defined ENaC sequence (see Fig. 1 B). A pre-M2 segment can arbitrarily be defined as a sequence delineated by a conserved Gly ( a G579) residue Portions of this work were previously published in abstract form (Kellenberger, S., N. Hoffmann-Pochon, E. Schneeberger, I. Gautschi, and L. Schild. 1998. J . Am . Soc . Nephrol . 9: A0193). Address correspondence to Laurent Schild, Institut de Pharmacologie et de Toxicologie, Bugnon 27, CH-1005 Lausanne, Switzerland. Fax: 4121 692 5355; E-mail: [email protected] 1 Abbreviations used in this paper: 3B2S, three-barrier–two-site; ENaC, epithelial Na 1 channel; I/V, current–voltage; K M , ion concentration for half-maximal unitary conductance; wt, wild type. on D ecem er 2, 2017 jgp.rress.org D ow nladed fom