Tsushima RG et al. (1997), Altered ionic selectivity of the sodium channel...

XB-ART-16712

J Gen Physiol 1997 Apr 01;1094:463-75.

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Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore.

Tsushima RG , Li RA , Backx PH .

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To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+ > Li+ > NH4+ > > Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+ and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+ > K+ > Na+ > or = Li+ approximately Cs+), D1532C, and G1533C (Na+ > Li+ > or = NH4+ > K+ > Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4+ > K+ > Na+ > or = Li+ approximately Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+ > or = Sr2+ > Mg2+ > Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.

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Species referenced: Xenopus laevis
Genes referenced: enah

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	Figure 2. Ion selectivity of the rSkM1 Na+ channel. (A) Whole-cell recordings of the rSkM1 Na+ channel in the presence of 96 mM NaCl, LiCl, NH4Cl, and KCl solutions. Oocytes were depolarized between −60 and +50 mV from a holding potential of −120 mV. Currents traces displayed are from −50 to +40 mV in 10-mV increments. Currents were corrected for leak and cell capacity. (B) Current-voltage relationship of the same oocyte shown in A for Na+ (▪), Li+ (○), NH4+ (♦), and K+ (▵). The data were fit to a Boltzmann distribution as described in materials and methods.
	Figure 3. Current ratios for (A) Li+, (B) NH4+, and (C) K+ of rSkM1 and cysteine mutant channels. Current ratios were determined from the ratio of the peak inward current in the presence of the test cation to the peak inward current in the presence of Na+. Data represent the mean ± SEM of three to five oocytes. n.e. denotes no expression. The P values are * < 0.05 and ** < 0.01.
	Figure 4. Altered ion selectivity of K1237C, W1531C, and D1532C. Whole-cell currents of (A) K1237C, (B) W1531C, and (C) D1532C recorded in a 96 mM NaCl or 96 mM KCl solution. Current recordings were measured from oocytes held at −120 mV and depolarized from −60 to +50 mV. Current traces shown are from −50 to +50 mV in 10-mV increments. Records were corrected for leak and capacitance current. The corresponding current-voltage relationship from the same oocytes shown below, recorded in Na+ (▪), NH4+ (▵), and K+ (○) solutions. Values were normalized to the maximal peak inward current for Na+. Data were fit to a Boltzmann distribution as described in Fig. 2.
	Figure 5. Current ratios for tryptophan mutants. Current ratios were calculated as described in Fig. 3. Values represent the mean ± SEM of five to seven oocytes. The P values are < 0.05, *< 0.01.
	Figure 6. Divalent permeation of K1237C. (A) Current recordings of K1237C in the presence of 96 mM Na+, Ca2+, Sr2+, Mg2+, and Ba2+ solutions. Currents were recorded as described in Fig. 4. (B) Corresponding current-voltage relationship from the same oocyte shown in A. Data were fit as described in Fig. 4. (C) Conductance-voltage relationship of K1237C in the presence of Na+ and the divalent cations. Symbols are the same as denoted in B. Data were normalized to the maximal Na+ conductance and fit to a Boltzmann distribution function. Values represent the mean ± SEM of five oocytes.
	Figure 7. Effect of MTS modification on the current-voltage relationship (left) and Cd2+ sensitivity (right) of (A) K1237C, (B) W1531C, and (C) D1532C. Currents were recorded as described in Fig. 4 in the absence and presence of 1 mM MTS derivative denoted. Fractional whole-cell currents (I/Io) measured at −10 mV were plotted as a function of Cd2+ concentration and fit to the Hill equation I/Io = 1/(1 + [Cd2+]/Kd) assuming a single binding site. The Kd for Cd2+ block for rSkM1 was 1,865 ± 386 μM for rSkM1 (n = 7). The Kd for Cd2+ block prior to and after modification by the MTS derivative was (A) K1237C 124 ± 22 μM (▪, n = 3) and 2,154 ± 182 μM (□, n = 3); (B) W1531C 55 ± 7 μM (○, n = 6) and 1,980 ± 176 μM (•, n = 3); (C) D1532C 583 ± 70 μM (▴, n = 6) and 2,325 ± 287 μM (▵, n = 3), respectively. Values represent the mean ± SEM.
	Figure 8. Effect of sulfhydryl modification of (A) K1237C, (B) W1531C, and (C) D1532C on ionic selectivity. Whole-cell currents were recorded in the presence of 96 mM NaCl or KCl solutions as described in Fig. 4. The corresponding current-voltage relationships of the cysteine mutants in the presence of Na+ (▪), K+ (○), and NH4+ (▴) are shown below. K1237C, W1531C, and D1532C were modified with 1 mM of MTSEA, MTSET, and MTSES, respectively.
	Figure 9. Effect of extracellular pH on rSkM1 and D1532C. (A) Effect of pH 5.6 (○), 7.6 (▪), and 9.6 (▵) on the current-voltage relationship of rSkM1 and D1532C. Currents were recorded as described in Fig. 4. (B) Effect of extracellular pH on the reversal potential (ENa) of rSkM1 (▪) and D1532C (○). Data represent the mean ± SEM of four to five oocytes. P values as in Fig. 3.

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Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase