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Figure 1. Sequence alignment of rat α, β, and γ subunits. The number of the start residue in the sequence is indicated. Residues mutated in this study are in bold. The region corresponding to the putative ENaC selectivity filter is shaded in gray.
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Figure 2. Single-channel properties of αβγ (wt) and αS592Tβγ channels with Na+ as the charge carrier. Cell-attached patch recordings were taken from oocytes expressing either wt-α or αS592T together with wt β and γ. (A) Exemplar traces of 100 s duration from patches containing wt or αS592Tβγ channels in the patch, with Na+ as the charge carrier. The pipet voltage was +110 mV. Downward deflections show inward current from the pipet to the cell from a fully closed (C) level to a number of open levels (O) as indicated. (B) All-points histograms were constructed from brief regions of the single-channel recordings obtained in A. The histograms were fit by either three (wt) or four (mutant) Gaussian functions corresponding to closed and open levels. Open conductance levels were of a single amplitude and no subconductances were observed. (C) Slope conductances of wt and αS592Tβγ channels were obtained from single-channel i-V relationships as shown. Conductance was calculated to be 4.2 ± 0.2 pS for the wt channel and 7.2 ± 0.2 pS for the mutant channel.
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Figure 3. Coexpression of wt and mutant αS592T subunits. Oocytes were injected with α-wt and αS592T subunits, together with wt β and γ. (A) Six sequential 10-s segments of recording from a patch containing wt, mutant, and hybrid channels are shown. The pipet contained Na+ and the pipet voltage was +110 mV. The wt and mutant conductance levels are indicated by the brackets to the left of the trace. Openings that correspond to a hybrid conductance are marked by a star. (B) An event-amplitude histogram was constructed by measuring the size of every closed to open transition (described in Materials and Methods) from the entire recording in A. The histogram was fit by three Gaussian functions where the parameters were (mean ± SD): â0.54 ± 0.03, â0.86 ± 0.04, and â0.73 ± 0.03 pA (marked by star), for the wt, mutant, and hybrid conductances, respectively.
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Figure 4. Single-channel properties of wt and αβG529Aγ channels with Li+ as the charge carrier. Cell-attached patch recordings were taken from oocytes expressing either wt or αβG529Aγ channels. (A) Exemplar single- channel traces of 60 s duration from patches containing wt or αβG529Aγ ENaC with LiCl in the pipet. The pipet voltage was +110 mV. Downward deflections correspond to movement of Li+ from the pipet to the cell from a fully closed (C) level to two open levels (O) as indicated. (B) All-points histograms were constructed from an â¼10-s region of the single-channel recordings obtained in A. The histograms were fit by three Gaussian functions corresponding to closed, one open, and two open levels. No subconductances were observed. (C) Slope conductances of wt and αβG529Aγ channels were obtained from single-channel i-V relationships as shown. Conductance was calculated to be 7.0 ± 0.1 pS for the wt channel and 1.9 ± 0.3 pS for the mutant channel.
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Figure 5. Coexpression of wt and mutant βG529A subunits. Oocytes were coinjected with β-wt and βG529A subunits, together with wt α and γ. (A) An 80-s segment of recording taken from a patch containing wt and mutant channels is shown. The pipet contained Li+ and the pipet voltage was +110 mV. The amplitude of wt and mutant conductance levels are indicated by brackets to the left of the trace. (B) An event-amplitude histogram was constructed by measuring the size of every closed to open transition for the recording with the most events shown in A (bottom trace). The histogram was fit by two Gaussian functions with parameters (mean ± SD): 0.28 ± 0.04 and 0.79 ± 0.05 pA, corresponding to mutant and wt conductance levels, respectively. Analysis of six other recordings gave similar results.
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Figure 6. Macroscopic current in oocytes expressing αβγG534E channels is inhibited by Ca2+. (A) Two-electrode voltage clamp was used to generate a doseâresponse curve showing the effects of increasing [Ca2+]ex on ILi in wt and αβγG534E channels. 100 μM amiloride was applied at the end of each recording to correct for amiloride-insensitive current. (B) Current is inhibited to a similar extent by 2 mM Ca2+ whether Li+ or Na+ is in the extracellular solution. *, statistically significant compared with current (ILi or INa) in the absence of Ca2+ (P < 0.05). Data presented as mean ± SEM for 7â12 oocytes.
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Figure 7. Single-channel properties of wt and αβγG534E channels. (A) Exemplar recordings taken from oocytes expressing wt channels with Li+ as the permeant ion, in the presence (+Ca) or absence (âCa) of 2 mM Ca2+ in the pipet as indicated. In each case, all-points histograms were constructed from â¼10-s regions of the trace above. The histograms were fit by three Gaussian functions corresponding to one closed and two open (O) levels as indicated. No subconductance levels were observed. (B) Exemplar recordings taken from oocytes expressing αβγG534E channels with Li+ as the permeant ion, in the presence (+Ca) or absence (âCa) of 2 mM Ca2+ in the pipet as indicated. Brief, large-amplitude transitions were noted during the recordings. We believe these are unlikely to represent ENaC and were ignored in the data analysis. For each trace, all-points histograms were constructed (from â¼10-s regions of recording) and fit by two Gaussian functions. No subconductance levels were observed. (C) Single-channel i-V relationship of wt channels with (+Ca) and without (âCa) 2 mM Ca2+ in the pipet. The gLi of wt channels with Ca2+ was 5.5 ± 0.2 pS and without Ca2+ was 7.0 ± 0.1 pS. (D) The single-channel conductance (gLi) of αβγG534E channels with Ca2+ was 3.4 ± 0.1 pS and without Ca2+ was 6.7 ± 0.1 pS.
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Figure 8. Coexpression of wt and mutant γG534E subunits. Oocytes were injected with γ-wt and γG534E subunits, together with wt α and β. (A) A continuous 80-s segment of a longer recording from a patch containing wt and mutant channels is shown. The pipet contained 110 mM Li+ and 2 mM Ca2+. The pipet voltage was 110 mV. The amplitude of wt and mutant conductance levels are indicated by brackets to the left of the trace. (B) An event-amplitude histogram was constructed by measuring the size of every open level transition for the full duration of the recording shown in A. The histogram was fit by two Gaussian functions with parameters (mean ± SD): 0.34 ± 0.04 and 0.76 ± 0.03 pA, corresponding to the mutant and wt conductance levels. Analysis of six other patches gave similar results.
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Figure 9. Single-channel properties of αS589Dβγ channels. Cell-attached patch recordings were taken from oocytes expressing αS589Dβγ channels. (A) An exemplar single-channel recording 60 s in duration from a patch containing an αS589Dβγ mutant. The pipet contained Li+ as the charge carrier and the pipet voltage was +110 mV. (B) An all-points histogram was constructed from the single-channel recording obtained in A. The histogram was fit by two Gaussian functions corresponding to a single closed and open conductance level. (C) The slope conductance of αS589Dβγ channels was obtained from the single-channel i-V relationship. Conductance was calculated to be 1.9 ± 0.1 pS for the mutant channel compared with 7.0 ± 0.1 pS for the wt channel.
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Figure 10. Coexpression of wt and mutant αS589D subunits. Oocytes were injected with α-wt and αS589D subunits, together with wt β and γ. (A) Four, 15-s segments of a longer recording containing mutant, wt, and hybrid channels in the patch are shown. Li+ was the charge carrier, and the pipet voltage was 110 mV. The brackets to the left of the traces indicate the approximate amplitude of the wt and mutant channels. Stars denote openings that correspond to a hybrid conductance. (B) An event-amplitude histogram was constructed from this recording and was fit by three Gaussian functions with parameters (mean ± SD): 0.28 ± 0.06, 0.86 ± 0.04, and 0.48 ± 0.05 pA, corresponding to the mutant, wt, and hybrid conductance levels, respectively.
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Figure 11. Coexpression of mutant αS589D and αS592T subunits. Oocytes were injected with αS589D and αS592T subunits, together with wt β and γ. (A) Four, 5-s segments of a longer recording containing mutant, wt, and hybrid channels in the patch are shown. The brackets to the left of the traces indicate the approximate amplitudes of wt and mutant channels. Li+ was the charge carrier, and the pipet voltage was 110 mV. Stars denote openings of a hybrid channel. (B) An event-amplitude histogram was constructed from this recording and was fit by three Gaussian functions with parameters (mean ± SD): 0.18 ± 0.03, 0.79 ± 0.04, and 0.61 ± 0.03 pA, corresponding to the αS589D, αS592T, and hybrid conductance levels, respectively.
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