Babini E et al. (2003), A new subunit of the epithelial Na+ channel ide...

XB-ART-5245

J Biol Chem 2003 Aug 01;27831:28418-26. doi: 10.1074/jbc.M301315200.

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A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition.

Babini E , Geisler HS , Siba M , Gründer S .

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The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.

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Species referenced: Xenopus laevis
Genes referenced: scnn1d snai1

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	FIG. 1. Sequence comparison of ENaC subunits. A, sequence alignment of xENaC with xENaC, xENaC, and xENaC. Amino acids showing a high degree of identity are shown as white letters on a black background. Transmembrane domains are indicated by bars, conserved cysteines by open circles, and putative N-linked glycosylation sites in the loop between transmembrane domains by a branched symbol. Transmembrane domains were predicted for xENaC with the TMpred program (available at www.ch.embnet.org/). GenBankTM/EBI accession numbers are AJ440222 for xENaC, U23535 for xENaC, U25285 for xENaC, and U25342 for xENaC. B, evolutionary relationship between ENaC subunits and other family members (FaNaCh, the FMRFamide receptor from the snail Helix aspersa; ripped pocket (RPK) and pickpocket (PPK) from Drosophila; acid-sensing ion channels (ASIC) from rat (r); and DEG-1, MEC-4, and MEC-10, degenerins from Caenorhabditis). Highly divergent sequences at the N and C termini as well as in the proximal part of the extracellular loop were deleted, and the alignment and the tree for the phylogram were established by neighbor joining with ClustalX. The tree was then imported in TreeView and rooted with DEG-1, MEC-4, and MEC-10 as an outgroup. h, human. x
	FIG. 2. Reverse transcription-PCR analysis of xENaC mRNA expression in different Xenopus tissues. PCR for glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) showed comparable amplification in all tissues examined. The control was without cDNA. The expression pattern was confirmed in two independent experiments.
	FIG. 3. Electrophysiological characteristics of xENaC. A, amplitude of amiloride (50 M)-sensitive currents in Xenopus oocytes injected with xENaC alone or with different subunit combinations (n 8). Oocytes were superfused with a solution containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES (pH 7.3); and amiloridesensitive current was measured using a two-electrode voltage clamp. The holding potential was 60 mV. B, amplitude of amiloride (Amil.)- sensitive currents of different subunit combinations with different extracellular cations (115 mM Na, Li, or K) at 60 mV (n 10). Low current amplitude with 2xENaC is also observed with xENaC-containing channels (14). C, dose-response relationship of amiloride blockade for xENaC and xENaC at 100 mV in a Na (115 mM)- containing solution. x
	FIG. 4. xENaC mediates Na self-inhibition. A, representative traces of inward currents after amiloride washout (50 M) for xENaC (black trace)- and xENaC (gray trace)-expressing oocytes. For better comparison, traces have been overlaid. B, left, representative traces of inward currents after amiloride washout with different Na concentrations (0, 3, 10, 35, 90, and 140 mM). Current traces have been normalized for channel rundown. Right, dependence on extracellular Na of the initial current amplitude for xENaC (open circles) and xENaC (closed circles) and of the current amplitude after 2 min (quasi-steady state) for xENaC (closed squares). C, representative traces of inward currents after switching from a solution of low (1 mM) to high (115 mM) Na concentration. D, self-inhibition is not due to an increase in the intracellular Na concentration. Channels were activated by washout with 50 M amiloride. The extracellular Na concentration was either 2 mM with a holding potential of 120 mV (gray trace) or 115 mM with a holding potential of 10 mV (black trace). Experiments were performed successively on the same oocyte. For better comparison, traces have been overlaid. E, BIG relieves inhibition by extracellular Na. Oocytes were superfused with a solution containing 1 mM NaCl, and channels were then “activated” by fast wash-in of a solution containing 115 mM NaCl. 1 mM BIG was applied after the current had relaxed to the steady state. xENaC a
	FIG. 5. Identification of molecular determinants of Na self-inhibition. Shown is a scheme of wild-type and chimeric subunits and representative current traces for each construct. All constructs were coexpressed with xENaC and xENaC. Oocytes were superfused with a solution containing 1 mM NaCl, and channels were then activated by fast wash-in of a solution containing 115 mM NaCl. Vertical and horizontal bars correspond to 1 A and 30 s, respectively. In construct C1, amino acids up to (not including) Thr49 of xENaC were replaced with the corresponding amino acids of xENaC; in C2, up to Gln68; in the C3 constructs, up to Pro258; in the C4 constructs, up to Met383; and in the C5 constructs, up to Leu510.
	FIG. 6. Identification of the amino acid responsible for the low apparent amiloride affinity of xENaC. Upper, sequence alignment of the second transmembrane domains of different ENaC subunits; lower, representative current traces of the xENaC mutant W513L after amiloride washout (left) or after switching from a solution of low (1 mM) to high (115 mM) Na concentration (right). The substitutioncontaining subunit was coexpressed with xENaC and xENaC. r, rat; h, human.