Schild L et al. (1997), Identification of amino acid residues in the al...

XB-ART-17228

J Gen Physiol 1997 Jan 01;1091:15-26.

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Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

Schild L , Schneeberger E , Gautschi I , Firsov D .

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The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.

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	Figure 2. Representative tracings of macroscopic inward Li+current recorded in three different oocytes expressing wild-type ENaC Î±Î²Î³ subunits. Substitution of 20 mM KCl with 20 mM LiCl in the external sucrose buffer medium (â¢) induced an inward Li+current that was inhibited by addition of 10 nM, 100 nM, 330 nM, 660 nM, 5 Î¼M indicated by the filled triangles (â´).
	Figure 3. Cell surface expression and macroscopic ILiof wt ENaC (n = 39), triple Cys mutant (Î±SCÎ²SCÎ³SC) (n = 36) and Î²Î³ subunits (n = 41). Filled bars represents ILi. Hatched bars represents specific binding per oocyte of M2AB ([125I]M2IgG1) antibodies (fmols/oocyte) directed against a FLAG epitope introduced in the ectodomain of Î² and Î³ wt and mutant subunits. *Denotes statistical significance <0.001.
	Figure 4. Effects of introducing Cys residues in pre M2 segment at positions Î±S583, Î²G525, and Î³G537. (A) Single channel tracings of Î²G525C mutant recorded with Na+as permeating ion. (B) Left: I-V relationships of wt ENaC and Î±S583C mutant. Cord conductance for wt ENaC (wt) in the presence of Na+was 5.1 and 10 pS with Li+. Li+conductance of Î±S583C was 8.9 pS. Right: I-V relationships for Î²G525C and Î³G537C. In the presence of Na+and Li+ions, cord conductances were, respectively, 3.2 and 5.8 pS for Î²G525C, 4 and 6.23 pS for Î³G534C. Dotted lines represent fit of the data of the I-V relationships according to the constant field equation. Each point represents mean value of two to five channels.
	Figure 5. Amiloride sensitivity of Î±Î²Î³ Cys mutants. (A) Original tracings of amiloride inhibition of inward Li+current in oocytes expressing Î²G525C mutant together with Î± and Î³ subunits. Triangles (â´) represent addition of external amiloride at increasing concentrations of 0.1, 10, 70, and 100 Î¼M, and â¢ indicates return to the starting K solution. (B) Titration curve of Li+current by amiloride. I/I0 is the ratio of blocked over unblocked Li+current. Li+ current was measured in individual oocytes in the presence of four different amiloride concentrations (wt-ENaC, n = 82; Î±S583C, n = 27; Î²G525C, n = 59; Î³G537C, n = 14) Dotted lines represent fit of data to a Langmuir inhibition isotherm (see materials and methods).
	Figure 6. Block of wt ENaC and mutants by external Zn2+. Concentration dependence of Zn2+ inhibition of inward Li+current. I/I0represents the current ratio measured in the presence/absence of external Zn2+. Inhibitory constants (Ki) were obtained from best fit of the data to a Langmuir inhibition isotherm (dotted lines). Zn2+ Ki were >10 mM for wt ENaC (n = 34), 29 Î¼M for Î±S583C (n = 42), 1.1 mM for Î²G525C (n = 10), and 1.9 mM for Î³G537C (n = 14).
	Figure 7. Single channel recording of Î±S583C mutant in absence or with 0.2 mM Zn2+in the external solution. In the presence of Zn2+ channel activity (n Â· Po) was 0.64 at â80 mV, 0.46 at â100 mV, 0.44 at â120 mV, and 0.35 at â150 mV.
	Figure 8. Effects on single channel conductance of introducing negatively charged residues Asp in the pre-M2 segment of Î±Î² subunits. (A) Upper tracing shows single channel recording in oocytes expressing Î±, Î²G522D, Î³ mutants. The lower tracings were recorded from an oocyte expressing both Î²G522D and Î² wild type together with Î± and Î³ subunits. Two channels with different conductances can be observed. (B) Left: I-V relationship of Î±S580D, Î²G522D, and Î²G522S in the presence of with 1.8 mM Ca2+ in the patch pipet. Cord Li+conductance was 9.6 pS for Î²G522S. Right: I-V relationship of S580D, G522D in the absence of Ca2+ in the pipet. Cord conductance were 6.7 and 11 pS for S580D in the presence of Na+and Li+respectively and 11 pS for Î²G522D with Li+ions. Each point represents a mean value of three to five channels.
	Figure 9. Block of Î±S580D, Î²G522D, Î³G534E mutants by external Ca2+. Mutant subunits were coexpressed with Î±, Î³ wild-type subunits. (A) Concentration dependence of inhibition of Li+current by external Ca2+. The ratio I/I0 represents the measured ILiin the presence/absence of external Ca2+ions. Ca2+ inhibitory constants (Ki) obtained from fit to inhibition Langmuir isotherm (dotted lines) were 2.1 mM for the Î±S580D (n = 8), 0.4 mM for Î²G522D (n = 10), and 2.2 mM for Î³G534E (n = 8). (B) Voltage dependence of Î²G522D mutant block by external Ca2+ at concentrations of 0.5, 1, and 2 mM (n = 4). Dotted lines represent fit of the data to Eq. 2 (see materials and methods) gave Ki(0) values of 0.67, 0.97, and 0.49, zâ² values of 0.26, 0.28, and 0.35 for the experiments done with respectively 0.5, 1, and 2mM Ca2+.
	Figure 10. Decreased sensitivity of Î²G522D mutant to amiloride. Dose-dependent inhibition of Li+current by amiloride. Inhibitory constants (Ki) obtained from fit of the data to inhibition Langmuir isotherm were 0.14 Î¼M for Î±S580D (n = 12), 0.06 Î¼M for Î³G534E (n = 8), 3.4 Î¼M for Î²G522D (n = 16), and 0.6 Î¼M for Î²G522S (nâ= 10).

References [+] :

Canessa, Membrane topology of the epithelial sodium channel in intact cells. 1994, Pubmed, Xenbase