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Figure 1. Alignment of the C-terminal transmembrane domains of human ASIC1a and rat ENaC subunits. The putative GAS selectivity region, named for the amino-acid sequence in ASIC1, is marked in blue, the conserved acidic amino acids in red, and the sites of Zn2+-sensitive cysteine locations in yellow. Bottom: The transmembrane segments in one subunit of the putative open structure of chicken ASIC (Baconguis et al., 2014; PDB accession no. 4NTW), and a cartoon representation of the transmembrane pore. The relevant elements in TM2 are indicated with the same color scheme.
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Figure 2. Current-voltage relationships in ENaC-expressing oocytes. (A) Currents in the presence of 110 mM extracellular Na+. (B) Currents in the presence of 110 mM extracellular K+. (C) I-V plots show currents as a function of voltage in the presence of Na+ with and without 10 µM amiloride, and the difference currents (INa). (D) Amiloride-sensitive currents in the presence of Na+ or K+.
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Figure 3. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with mutations in the E18′ positions. (A) WT. (B) αE595Q/β/γ. (C) α/βE537Q/γ. (D) α/β/γE549Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+ (I0). Data represent means ± SEM for four to seven oocytes.
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Figure 4. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with mutations in the E21′ positions. (A) WT. (B) αE598Q/β/γ. (C) α/βE540Q/γ. (D) α/β/γE552Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+ (I0). Data represent means ± SEM for three to seven oocytes.
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Figure 5. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with truncations in the β subunit. (A) α/βΤ/γ. (B) αE595Q/βΤ/γE549Q. (C) αE598Q/βΤ/γE552Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to six oocytes.
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Figure 6. Current-voltage relationships in the presence of 110 mM Na+ and 110 mM K+ in oocytes expressing ENaC with truncations in the β subunit and αC506S mutations. (A) WT. (B) αC506S/βΤ/γ. (C) αC506S E595Q/βΤ/γ. (D) αC506S/βΤE537Q/γE549Q. All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to six oocytes.
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Figure 7. Single-channel currents in E18′Q mutants. Currents were recorded in the cell-attached mode at a pipette potential of +100 mV with 110 mM LiCl in the pipette. (A) WT ENaC. (B) αE598Q/β/γ. (C) α/β/γE552Q. (D) Single-channel i-V relationships for the channels shown in A through C. Data represent means ± SEM for two to seven patches. Slopes estimated by linear regression gave conductances of 6.9 ± 0.6 pS (WT), 7.6 ± 0.2 pS (αE598Q/β/γ), and 8.9 ± 0.2 pS α/β/γE552Q. The last value is significantly greater than that of the WT channel (P = 0.04).
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Figure 8. Voltage dependence of block by K+ in oocytes expressing ENaC. (A) INa-V relationships in 20 mM NaCl with 90 mM NMDG+ and no K+, or 90 mM K+. (B) Plot of ln(I0/IK – 1) versus voltage where I0 and IK are currents in the absence and presence of K+, respectively. The slope of the relationship obtained by linear regression gives KK(0) = 450 ± 70 mM, z•δ = 0.20 ± 0.03 (n = 5).
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Figure 9. Voltage dependence of block by guanidinium in oocytes expressing ENaC. (A) INa-V relationships in 30 mM NaCl with 80 mM NMDG+ and no guanidinium or 80 mM guanidinium. (B) Plot of ln(I0/Igdm – 1) versus voltage where I0 and Igdm are currents in the absence and presence of guanidinium, respectively. The slope of the relationship obtained by linear regression gives Kgdm (0) = 320 ± 40 mM, z•δ = 0.24 ± 0.02 (n = 6).
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Figure 10. Effect of Na+ on block by K+ in oocytes expressing ENaC. (A) INa-V relationships in 10 mM Na+ with and without 70 mM K+. (B) INa-V relationships in 40 mM Na+ with and without 70 mM K+. (C) KK at Vm = −150 mV with 10 and 40 mM Na+. Values are significantly different (P = 0.002). (D) KK and KNa as a function of voltage.
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Figure 11. Voltage dependence of block by Zn2+ in oocytes expressing αS583C/β/γ ENaC. (A) INa-V relationships for WT ENaC in 110 mM NaCl with no Zn2+ and 0.2 mM Zn2+. (B) INa-V relationships for ENaC αS583C/β/γ in 110 mM NaCl with no Zn2+ and 0.2 mM Zn2+. (C) Plot of ln(I0/IZn – 1) versus voltage, where I0 and IZn are currents in the absence and presence of Zn2+, respectively, for αS583C/β/γ. The slope of the relationship obtained by linear regression gives KZn(0) = 230 ± 40 µM, z•δ = 0.14 ± 0.01 (n = 5), assuming no stimulation of the channel by Zn2+ (red). Analysis assuming the same activation of WT and αS583C/β/γ gives KZn(0) = 150 ± 20 µM, z•δ = 0.06 ± 0.01 (black).
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Figure 12. Voltage dependence of block by amiloride in oocytes expressing WT and αS583C ENaC. (A) INa-V relationships for WT in 20 mM NaCl with no amiloride or 0.1 µM amiloride (amil). (B) Plot of ln(I0/Iamil – 1) versus voltage where I0 and Iamil are currents in the absence and presence of amiloride, respectively. The slope of the relationship obtained by linear regression gives Kamil = 64 ± 11 nM, z•δ = 0.03 ± 0.01 (n = 6). (C) INa-V relationships for αS583C in 110 mM NaCl with no amiloride or 10−6 M amiloride. (D) Plot of ln(I0/Iamil – 1) versus voltage. The slope of the relationship obtained by linear regression gives Kamil = 2.0 ± 0.2 µM, z•δ = 0.12 ± 0.01 (n = 4).
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Figure 13. Current-voltage relationships in WT ENaC and ENaC(GAS)3. (A and B) Currents were measured in the presence of 110 mM Na+, 110 mM K+, 110 mM guanidinium+, and 110 mM Li+ in oocytes expressing ENaC (A) and ENaC(GAS)3 (B). All data are corrected for amiloride-insensitive currents and normalized to the value at Vm = −100 mV in the presence of Na+. Data represent means ± SEM for three to eight oocytes.
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Figure 14. Single-channel currents in WT ENaC and ENaC(GAS)3. Currents were recorded in the cell-attached mode at a pipette potential of +100 mV with 110 mM NaCl or 110 mM LiCl in the pipette. Top: Typical traces from WT and ENaC(GAS)3. Bottom: i-V relationships. Data represent means ± SEM for three to five patches. Slope conductances for WT were 5.3 pS for Na+ and 6.9 pS for Li+. For ENaC(GAS)3, they were 1.6 pS for Na+ and 8.1 pS for Li+.
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