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Figure 1. Effect of extracellular Iâ on hClC-1 expressed in Xenopus oocytes. (A) Current responses to voltage steps from a holding potential of â30 mV in 40-mV steps from â145 to +15 mV measured in standard extracellular solution. Each test pulse is followed by a step to â125 mV. (B) Current responses to the same pulse protocol in the same oocyte as in A after substitution of 96 mM NaCl by an equimolar concentration of NaI. (C) Voltage dependence of the instantaneous current amplitude for different extracellular [Iâ]. Data represent mean ± SEM from four different cells (every cell was tested with each [Iâ]). Current amplitudes were normalized to the instantaneous current amplitude measured at â145 mV in the absence of extracellular Iâ. (D) Voltage dependence of the late current amplitude obtained from the same data set shown in C. Data were normalized in the same way as in C.
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Figure 2. Effect of other extracellular anions on hClC-1 expressed in Xenopus oocytes. Current responses to voltage steps between â125 and +35 mV in 40-mV steps from a holding potential of â30 mV from a single oocyte are shown. In A, the extracellular solution was ND-96. For the three other recordings, 48 mM NaCl was substituted by an equimolar concentration of NaCH3SO3 (B), NaNO3 (C), and NaSCN (D).
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Figure 3. Normalized currentâvoltage relationships for hClC-1 in various extracellular solutions. For each anion tested, 48 mM NaCl was replaced by equimolar NaX, with X denoting different anions. For each anion, instantaneous (A and C) and steady state (B and D) currents were measured during voltage steps from a holding potential of â30 mV and normalized to the instantaneous current amplitude at â145 mV for the same oocyte in ND-96. Each point represents mean ± SEM for at least three cells. (A) Voltage dependence of the instantaneous current amplitude for extracellular solutions containing 96 mM NaCl (â¢), 48 mM NaCl + 48 mM NaBr (â¡), 48 mM NaCl + 48 mM NaSCN (â´), and 48 mM NaCl + 48 mM NaNO3 (â¿). (B) Voltage dependence of the steady state amplitudes from the same recordings as shown in A. (C) Voltage dependence of the instantaneous current amplitude for extracellular solutions containing 96 mM NaCl (â¦), 48 mM NaCl + 48 mM Na-gluconate (â), 48 mM NaCl + 48 mM Na-cyclamate (â¢), and 48 mM NaCl + 48 mM NaCH3SO3 (â¡). (D) Corresponding steady state values from the experiment shown in C. (E) Correlation between a blocking parameter (Ipeak in the presence of 48 mM anion divided by Ipeak in the presence of Clâ measured at â145 mV) and relative late current (Ilate divided by Ipeak measured at â145 mV) for various extracellular anions.
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Figure 4. Effect of extracellular Iâ on hClC-1 deactivation. (A) Voltage dependence of fast and slow time constants in the absence (filled symbols) or presence (open symbols) of 40 mM Iâ. (BâD) Voltage dependence of the fractional amplitudes A1 (B), A2 (C), and C (D) determined in the presence of three different extracellular Iâ concentrations. Extracellular [Iâ] was increased by replacing NaCl with equimolar amounts of NaI in the external solution. Each point represents mean ± SEM for at least three cells.
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Figure 5. Relationship between extracellular Iâ concentration and fractional current amplitudes. Fractional current amplitudes were determined at three different test potentials (â75, â115, and â165 mV) as described in methods. Solid lines represent fitted hyperbola (see text) to the measured data. Each point represents mean ± SEM for at least three cells. (A) Fast deactivating fractional current amplitude, A1. (B) Slow deactivating fractional current amplitude, A2. (C) Nondeactivating fractional current amplitude, C. Insets show transformations of the data with fitted regression lines as described in results. Hill coefficients determined from the regression lines are as follows: A1: 0.47, 0.96, and 1.00; A2: 0.59, 0.98, and 0.22; C: 0.31, 0.86, and 0.99 for test potentials of â75, â115, and â155 mV, respectively.
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Figure 6. Voltage dependence of Iâ binding in hClC-1. Voltage dependence of the apparent dissociation constants (KD) for the three different kinetic states shown as linear (A) and semi-log (B) plots. Lines indicate fits of the Woodhull formula: KD = KD(0 mV) eâFδV/RT, where KD(0 mV) denotes the estimated value at a membrane potential of 0 mV, and δ is the electrical distance measured from the extracellular side of the membrane. (C) Voltage dependence of the limiting values for the derived fractional current amplitudes at very high extracellular Iâ concentration (pI). (D) Voltage dependence of the limiting values for the derived fractional current amplitudes at zero extracellular [Iâ] (po).
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Figure 8. Effect of intracellular Iâ on hClC-1 current in HEK-293 cells. (A) Current responses to voltage steps to â165 and +55 mV from a holding potential of 0 mV. Data shown are from three different cells exposed to three different internal Iâ concentrations as indicated in the figure. Currents were normalized to the current amplitude measured at +55 mV. (B) Voltage dependence of the instantaneous current amplitude for different intracellular Iâ concentrations. Iodide concentration was changed by replacing different amounts of Na-gluconate with NaI in a pipette solution containing (mM): 50 NaCl, 80 Na-gluconate, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4. Each point represents mean ± SEM from four cells. Current amplitudes were normalized to the value measured at +75 mV.
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Figure 9. Effect of different intracellular anions on hClC-1 stably expressed in HEK-293 cells. (A, C, and E) Current responses to voltage steps from a holding potential of 0 mV to test potentials between â165 and +75 mV in 80-mV steps. Each step is followed by a test potential of â85 mV. Cells were bathed in standard extracellular solution and perfused intracellularly with a solution containing (mM): 50 NaCl, 50 NaX, 30 Na-gluco-nate, 2 MgCl2, 5 EGTA, 10 HEPES where X denotes Iâ (A), NO3â (C), or SCNâ (E). (B, D, and F) Voltage dependence of the instantaneous current amplitudes from recordings shown in A, C, and E. (G) Correlation of the potency to block Clâ currents from the intracellular site and the fast deactivation time constant Ï1 measured at a test potential of â145 mV. We defined a blocking parameter by dividing the current amplitude measured at +55 mV (which is not affected by intracellular anions) by the amplitude at â145 mV for each cell. Data points represent mean ± SEM from three cells.
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Figure 10. Effect of intracellular Iâ on deactivation time constants. (A) Voltage dependence of the fast deactivation time constants (Ï1) for different intracellular Iâ concentrations. Each point represents mean ± SEM from four cells. (B) Voltage dependence of the slow deactivation time constants (Ï2) for different intracellular Iâ concentrations.
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Figure 11. Intracellular Iâ concentration dependence of hClC-1 deactivation. Reciprocal deactivation time constants determined at six different test potentials in the presence of different intra-cellular Iâ concentrations. (A) Iâ concentration dependence of the fast deactivation time constant (Ï1). (B) Iâ concentration dependence of the slow deactivation time constant (Ï2). In A and B, solid lines represent fitted hyperbola to the measured data (see text), and each point represents mean ± SEM for at least three cells. (C) Voltage dependence of KD determined from data fits shown in A and B. (â¢) Fast deactivating state, (â¡) slow deactivating state. Lines indicate Woodhull fits: KD = KD(0 mV) eFδV/RT, where KD(0 mV) denotes the estimated value at a membrane potential of 0 mV and δ the electrical distance measured from the extracellular side of the membrane. (D) Voltage dependence of Ïmin for the derived values for fast (Ï1, â¢) and slow (Ï2, â¡) deactivation time constants.
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Figure 12. Concentration dependence of the reversal potential and mole fraction behavior in hClC-1. (A) Dependence of the reversal potential on extracellular Clâ concentration measured in HEK-293 cells. Current reversal potentials were measured under biionic conditions with Iâ as the only permeant intracellular and Clâ as the only permeant extracellular anion. Iodide and Clâ concentrations were changed proportionally. (B and C) Mole fraction behavior of hClC-1 instantaneous current amplitudes measured in Xenopus oocytes at â145 and â85 mV for mixtures of Clâ and Iâ (B), and Clâ and SCNâ (C). Instantaneous current amplitudes were measured on three different cells For each oocyte, five different mole fractions were tested. The mole fraction was changed by replacing equimolar amounts of NaCl with either NaI or NaSCN. Relative current was calculated by normalizing instantaneous current amplitudes to the value obtained at â145 mV measured in ND-96 solution.
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