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Figure 1. Effect of extracellular I− on hClC-1 expressed in Xenopus oocytes. (A) Current responses to voltage steps from a holding potential of −30 mV in 40-mV steps from −145 to +15 mV measured in standard extracellular solution. Each test pulse is followed by a step to −125 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 [I−]. Data represent mean ± SEM from four different cells (every cell was tested with each [I−]). Current amplitudes were normalized to the instantaneous current amplitude measured at −145 mV in the absence of extracellular I−. (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 −125 and +35 mV in 40-mV steps from a holding potential of −30 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 current–voltage 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 −30 mV and normalized to the instantaneous current amplitude at −145 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 (•), 48 mM NaCl + 48 mM NaBr (□), 48 mM NaCl + 48 mM NaSCN (▴), and 48 mM NaCl + 48 mM NaNO3 (▿). (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 (♦), 48 mM NaCl + 48 mM Na-gluconate (○), 48 mM NaCl + 48 mM Na-cyclamate (•), and 48 mM NaCl + 48 mM NaCH3SO3 (□). (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 Cl− measured at −145 mV) and relative late current (Ilate divided by Ipeak measured at −145 mV) for various extracellular anions.
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Figure 4. Effect of extracellular I− 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 I−. (B–D) Voltage dependence of the fractional amplitudes A1 (B), A2 (C), and C (D) determined in the presence of three different extracellular I− concentrations. Extracellular [I−] 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 I− concentration and fractional current amplitudes. Fractional current amplitudes were determined at three different test potentials (−75, −115, and −165 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 −75, −115, and −155 mV, respectively.
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Figure 6. Voltage dependence of I− 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) e−FδV/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 I− concentration (pI). (D) Voltage dependence of the limiting values for the derived fractional current amplitudes at zero extracellular [I−] (po).
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Figure 8. Effect of intracellular I− on hClC-1 current in HEK-293 cells. (A) Current responses to voltage steps to −165 and +55 mV from a holding potential of 0 mV. Data shown are from three different cells exposed to three different internal I− 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 I− 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 −165 and +75 mV in 80-mV steps. Each step is followed by a test potential of −85 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 I− (A), NO3− (C), or SCN− (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 Cl− currents from the intracellular site and the fast deactivation time constant τ1 measured at a test potential of −145 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 −145 mV for each cell. Data points represent mean ± SEM from three cells.
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Figure 10. Effect of intracellular I− on deactivation time constants. (A) Voltage dependence of the fast deactivation time constants (τ1) for different intracellular I− concentrations. Each point represents mean ± SEM from four cells. (B) Voltage dependence of the slow deactivation time constants (τ2) for different intracellular I− concentrations.
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Figure 11. Intracellular I− concentration dependence of hClC-1 deactivation. Reciprocal deactivation time constants determined at six different test potentials in the presence of different intra-cellular I− concentrations. (A) I− concentration dependence of the fast deactivation time constant (τ1). (B) I− concentration dependence of the slow deactivation time constant (τ2). 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. (•) Fast deactivating state, (□) 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 τmin for the derived values for fast (τ1, •) and slow (τ2, □) 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 Cl− concentration measured in HEK-293 cells. Current reversal potentials were measured under biionic conditions with I− as the only permeant intracellular and Cl− as the only permeant extracellular anion. Iodide and Cl− concentrations were changed proportionally. (B and C) Mole fraction behavior of hClC-1 instantaneous current amplitudes measured in Xenopus oocytes at −145 and −85 mV for mixtures of Cl− and I− (B), and Cl− and SCN− (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 −145 mV measured in ND-96 solution.
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