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FIGURE 1. Location of residue Lys210 mapped on the structure of the CLC-ec1 mutant E148Q (PDB entry 1OTU). A, dimeric structure of CLC-ec1 viewed from the membrane plane (extracellular side above and cytoplasmic side below). The two subunits are shown in green and cyan. Residue Gln148 is colored in red, Arg147, corresponding to Lys210 of CLC-5, is shown in blue, and Ser107 in orange. Chloride anions bound to Sext, Scen, Sint are shown in magenta. B, expanded representation of the anion permeation pathway for one of the subunits. The positions of the three binding sites are also indicated by horizontal dashed lines. C, alignment of the sequence stretch comprising Lys210 (shown in bold) for several CLC proteins.
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FIGURE 2. Expression and rectification of the currents for WT CLC-5 and Lys210 mutants. A, representative current recordings for WT and the indicated mutants. B, average of the ratio between the current of WT and the indicated mutants at 100 mV. In each batch of oocytes, the ratio was calculated by dividing the average current of the mutants and the WT. Data were obtained for at least three batches (n ≥ 7). Error bars are S.D. The average current of the WT was 5.7 ± 1.8 μA (n = 38). C, average values of the ratio of currents at 120 and 80 mV calculated for each individual oocyte for WT (Lys210) and the indicated mutants (the number of experiments is the same as in panel B, error bars are S.D.). The difference with the WT value is statistically significant for K210R (p < 10−6) and K210C and K210E (p < 0.05).
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FIGURE 3. Selectivity of WT CLC-5 and Lys210 mutants. A, representative current recordings in Cl− solution (black trace) and NO3− solution (red trace) at 100 mV for two oocytes expressing WT CLC-5 and the mutant K210A, respectively. The ratio INO3−/ICl− was 1.61 for WT and 0.67 for K210A. Traces for K210A were scaled to obtain the same current level in Cl− solution for WT and K210A. B, average currents in Br−, NO3−, and I− relative to Cl− at 100 mV (n ≥ 7). The difference with the WT value of INO3−/ICl− is statistically significant for all the mutants (p < 0.01), for IBr−/ICl− and IIodide−/ICl− is statistically significant only for K210H (p < 0.05). C, average of the currents in SCN− relative to Cl− at 80 mV (n ≥ 7). The difference with the WT value is statistically significant for K210A, K210E, and K210Q (p < 0.05).
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FIGURE 4. Dependence of WT and K210A on [Cl−]ext. A, dependence on [Cl−]ext of the normalized currents for WT (n = 5, filled squares) and K210A (n = 3, open circles) at 100 mV (error bars are smaller than symbols). Normalization was performed with the current at 100 mm. Curves are fits to Equation 2, with K1/2 as the fitted parameter resulting in values of 71 ± 6 and 116 ± 10 mm for WT and K210A, respectively. B, voltage dependence of K1/2. Symbols are the same as panel A. Lines are fit with Equation 4 with K(0) and z as the fitted parameters resulting in values of 1064 ± 27 and 0.67 ± 0.09 mm for WT and 860 ± 15 and 0.50 ± 0.06 mm for K210A, respectively.
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FIGURE 5. Proton transport properties of WT and K210A. A, representative recordings of the extracellular acidification produced by the activation of WT and K210A. Horizontal gray bars indicate stimulation with trains of voltage pulses to 80 mV (400 ms duration) in Cl− (filled squares) and NO3− (open circles) solutions. B, average coupling efficiency of Cl− and NO3− with H+ transport, erel (Equation 8), at 80 and 100 mV measured for WT (n = 4) and K210A (n = 6) (errors bars are S.D.). The difference is not statistically significant (p > 0.05).
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FIGURE 6. MTSET modification of K210C. A, representative current recordings showing the effect on the stationary current level of 1 mm MTSET (application indicated by a black bar) and of the washout with 10 mm DTT (indicated by a gray bar). Both reagents were dissolved in measuring solution containing 100 mm NO3−. Voltage protocol consisted of a 20-ms pulse to 80 mV applied every 2 s. After MTSET application the experiment was interrupted to record I-V relationships in Cl− and NO3− solutions. The dashed line represents the 0 current level. B, representative current recordings in Cl− (solid trace) and NO3− (dashed trace), at 100 mV from the same oocyte as shown in panel A (before and after MTSET application and after washout with DTT). In these conditions the value of INO3−/ICl− was, respectively, 0.89, 1.50, and 0.91. C, average value of INO3−/ICl− before and after MTSET application (n = 15) and after DTT washout (n = 6). The increase of INO3−/ICl− after MTSET modification is statistically significant (p < 10−6).
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FIGURE 7. MTSEH modification of K210C. A, representative current recordings showing the effect of 1 mm MTSEH on the stationary current level followed by application of 1 mm MTSET. Both reagents were dissolved in measuring solution containing 100 mm NO3−. Application of MTSEH is indicated by a gray bar and application of MTSET by a black bar. After MTSEH application the experiment was interrupted to record I-V relationships in Cl− and NO3− solutions. The dashed line represents the 0 current level. B, representative current recordings in Cl− (solid trace) and NO3− (dashed trace), at 100 mV from the same oocyte shown in panel A (before and after MTSEH application and after application of MTSET). In these conditions the value of INO3−/ICl− was, respectively, 0.91, 0.91, and 0.88. C, average value of INO3−/ICl− before and after MTSEH application (n = 6) and after application of MTSET (n = 3). The differences of the values of INO3−/ICl− in these three different conditions were not statistically significant (p > 0.8).
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FIGURE 8. Dependence of WT and K210A on [NO3−]ext and voltage. A, dependence on [NO3−]ext of the normalized currents for WT (n ≥ 7, filled squares) and K210A (n ≥ 8, open circles) at 120 mV. Normalization was performed with the current in 100 mm Cl− at 120 mV. Solid and dashed curves are fits to Equation 3, with K1/2 and ImaxNO3−/ImaxCl− as the fitted parameters resulting in values of 171 ± 26 and 372 ± 74 for K1/2 and 2.9 ± 0.2 and 2.0 ± 0.2 mm for ImaxNO3−/ImaxCl−, for WT and K210A, respectively. B, voltage dependence of K1/2. Symbols are the same as in panel A. Lines are fit with Equation 4 with K(0) and z as the fitted parameters resulting in values of 3.2 ± 0.6 and 0.74 ± 0.07 m for WT and 11.9 ± 3.4 and 0.62 ± 0.05 m for K210A, respectively.
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