Kuruma A, Hartzell HC.

R01GM55276 NIGMS NIH HHS

	Scheme S1.
	Figure 2. Excised patch currents are Cl− currents. The reversal potential of the Ca2+-activated currents recorded in inside-out patches was determined by measuring the instantaneous current at different potentials following a depolarizing step to +120 mV (voltage protocol is shown above B). The pipet solution contained either 160 (A) or 40 (B) mM Cl−. The bath solution contained 160 mM Cl−. (C) Instantaneous current–voltage relationship. The amplitudes of the tail currents were plotted versus the membrane potential for symmetric 160 mM Cl− (○) or for 40 mM Clo–160 mM Cli (•). The reversal potential shifted from 0 to +38.1 mV with the reduction in extracellular Cl. The shift for a Cl-selective channel predicted by the Goldman-Hodgkin-Katz equation is +35.2 mV.
	Figure 1. Activation of Ca2+-activated Cl− currents in an excised inside-out patch from a Xenopus oocyte. The cytosolic face of an excised inside-out patch was exposed to NMDG-Cl solutions containing <10 nM (A and C) or 600 nM (B) Ca2+. The patch was voltage clamped by stepping from a holding potential of 0 mV to various potentials between +120 and −120 mV for 1.3 s, followed by a 0.3-s step to −120 mV (voltage protocol is shown above B). The largest outward current corresponds to the +120-mV pulse. (D) Steady state current–voltage relationship for excised patch current. The currents at the end of the 1.3-s pulse from B were plotted versus membrane potential. •, 600 nM Ca2+; ○, ,10 nM Ca2+. (E–F) Comparison of currents in excised patch with whole-cell currents. (E) The “excised-patch” current was recorded with symmetrical Cl− at a transmembrane voltage of +200 mV. The cytosolic face of the patch was exposed to (a) <10 nM, (b) 460 nM, (c) 1. 1 μM, and (d) 1.8 μM Ca2+. (F) The “whole-cell” current (ICl1-S) was recorded in an intact oocyte by two-electrode voltage clamp after injection of (a) none, (b) 320 pmol Ca2+ (10 s after injection), (c) 690 pmol Ca2+ (10 s after injection), and (d) 690 pmol Ca2+ (20 s after injection). The transmembrane voltage was +80 mV and extracellular Cl− was 134 mM. Tail currents for both E and F were recorded at −120 mV.
	Figure 3. Ca2+ dependence of Cl− currents in a single excised patch. The cytosolic face of an excised patch was exposed to solutions with different free [Ca2+]: A, <10 nM Ca2+; B, 70 nM Ca2+; C, 600 nM Ca2+; D, 1 μM Ca2+; E, 2 μM Ca2+; and F, <10 nM Ca2+ (after washing out 2 μM Ca2+). The patch was voltage clamped by stepping to various potentials between +120 and −120 mV for 1.3 s from the holding potential of 0 mV, followed by a step to −120 mV for 0.3 s (protocol shown above B). (G) Steady state current–voltage relationships of currents at 600 nM Ca2+, 1 μM Ca2+, and 2 μM Ca2+.
	Figure 4. Voltage dependence of Ca2+-dependent conductance. The voltage protocol was the same as in Fig. 3, but conductance was calculated by dividing the tail currents at −120 mV by the driving force, and then normalizing the data to the maximum conductance at +120 mV at 1.1 μM Ca2+ (∼0.5 nS). (A) Average of experiments at 2.2 μM Ca2+ (n = 5), 1.1 μM Ca2+ (n = 4), 540 nM Ca2+ (n = 13), 300 nM Ca2+ (n = 12), 190 nM Ca2+ (n = 5), and 70 nM Ca2+ (n = 11). The solid curves are fits to the Boltzmann equation. (B) Plot of the estimate of V1/2 vs. [Ca2+]. □, typical patch; •, averages from A.
	Figure 5. Hyperpolarization cannot turn off currents activated by Ca2+. Excised patches were exposed to 500 nM Ca2+ (A and B) or 1 μM Ca2+ (C and D). The membrane potential was held at various values between −200 and 0 mV. The instantaneous currents at +120 mV were measured to determine the conductance activated at the preceding voltage. At both [Ca2+], hyperpolarization to −200 mV was not able to inactivate the Ca2+-activated current. Only selected traces are shown in A and C for clarity.
	Figure 6. Voltage-dependent Ca2+ affinity of Ca2+-activated Cl channels. (A) Time course of current rundown in a champion patch. The amplitude of currents at +120 mV in the presence of different [Ca2+] are plotted with time after patch excision. The patch was exposed to Ca2+ only during the voltage-clamp episodes. (B) Voltage-dependent conductance of the champion patch. The experiment was performed as described in Fig. 4 A. (C) Voltage dependence of Ca2+ affinity of champion patch. The tail current amplitudes used to create the plot in B were replotted as a function of [Ca2+] and fitted to the Hill equation. (D) The best-fit parameters of the data in C to the Hill equation. (E) Demonstration that 40 μM Ca2+ produces a maximal Cl current. Steady state I-V curves are shown for Ca2+-dependent currents in 40 μM (•) and ∼900 μM (▪) Ca2+. (F) Average apparent affinity of the channel for Ca2+ at different voltages. Normalized conductance from Fig. 4 A was replotted as a function of [Ca2+]. Error bars are not shown for clarity but can be obtained from Fig. 4. The data for [Ca2+] < 2 μM were fitted to the Hill equation.
	Figure 7. Kinetic analysis of deactivation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was bathed in solutions with different free [Ca2+]: (A) 280 nM Ca2+, (B) 460 nM Ca2+, (C) 670 nM Ca2+, (D) 1.1 μM Ca2+, and (E) 1.8 μM Ca2+. The patch was voltage clamped by depolarizing to +120 mV, and then stepping to various potentials between +120 and −120 mV. The tail currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential. Solid curves in the right panels are best fits of the solid symbols to the equation τdeact = Ae q FV/RT + b.
	Figure 8. Average data for deactivation of Ca2+-activated Cl− currents. The experiments were performed as in Fig. 7. (A) The data were averaged for 280 nM Ca2+ (n = 7), 460 nM Ca2+ (n = 6), 680 nM Ca2+ (n = 5), and 1.1 μM Ca2+ (n = 5). The data were fitted to the equation τdeact = Ae q FV/RT + b. (B) The q values calculated from the fits in A were plotted vs. [Ca2+].
	Figure 13. Anionic selectivity of the channel. The reversal potential of the outward currents activated by 1 (A–C) and 2 (D–F) μM Ca2+ were determined by measuring the instantaneous tail currents at different potentials following a pulse to +100 mV. The voltage protocol is shown above A. The reversal potential of the inward currents activated by 2 μM Ca2+ (G–I) were determined in the same way following a pulse to −100 mV. The voltage protocol is shown above G. The pipet solution contained 158.4 mM Cl−, the bath contained either 158.4 mM Cl− (A, D, and G) or 150 mM I− and 8.4 mM Cl− (B, E, and H). (C, F, and I) Tail current amplitudes for the symmetrical chloride solutions (•) and the low Cl−, high iodide solution (○). (J) Bar graph of the anionic permeability ratios (Px/PCl) for outward currents at 1 and 2 μM Ca2+ and for inward current at 2 μM Ca2+.
	Figure 9. Kinetic analysis of activation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was exposed to solutions with different free [Ca2+]. The patch was voltage clamped by stepping to various potentials between +200 and +40 mV. The activating phase of the currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential.
	Figure 10. Average data for activation of Ca2+-activated Cl− currents in excised patch. The experiments were performed as in Fig. 9. (A) Dependence of current activation on voltage at different [Ca2+]. (B) The data from A were replotted to show the dependence of current activation on free [Ca2+].
	Figure 11. Activation of Ca2+-activated Cl− currents in an excised patch by rapid perfusion of Ca2+. (A) Calibration of rate of change of solution. The liquid junction potential of a high resistance (50 MΩ) electrode placed in the solution stream was measured as the solution was changed from 0.1 to 2 M KCl. (Top) Solenoid voltage, (bottom) liquid junction potential. There is an ∼40-ms lag between switching the solenoid and the onset of the change in junction potential due to the dead volume of the perfusion line. Once the potential begins to change, the time to change from 10 to 90% of maximum was ∼3 ms. (B) Protocol. The patch was switched to a voltage between +120 and −120 mV from the holding potential of 0 mV, 5 s before changing the perfusion from low to high [Ca2+]. (C) Current traces recorded upon switching from <10 nM Ca2+ to 40 μM Ca2+ (the activation and deactivation are fitted to single exponentials; superimposed). (D) The time constant of turn off (τoff) of the current was plotted versus potential. (E) The time constant of the turn on (τon) of the current was plotted versus potential. (F) Current traces recorded upon switching from <10 to 450 nM Ca2+. Single exponential fits to activation and deactivation are superimposed. (G and H) Time constants as a function of potential.
	Figure 12. Average data (n = 3–15) from rapid perfusion experiments. Experiments were performed as described in Fig. 11. τoff (A) and τon (B) are plotted for 40 μM Ca2+ (□), 1 μM Ca2+ (▴), 630 nM Ca2+ (○), 400 nM Ca2+ (♦). The solid line in A is a single exponential fit to the average of all the data at different [Ca2+]: τoff = 0.064 * exp (0.26 * FV/RT).
	Figure 14. Simulation of Ca2+-activated Cl currents using the model and rate constants described in the text. The macroscopic currents were modeled using a Monte-Carlo simulation program written by Dr. Steve Traynelis. (A and B) Simulated currents in response to Ca2+ steps from <10 nM to 50 μM (A) or 500 nM (B). (C and D) Simulated currents in response to voltage-clamp pulses from 0 mV to voltages between +120 and −120 mV (20-mV increments) at a steady 50 μM Ca2+ (C) or 500 nM Ca2+ (D). (E) Deactivation time constants measured from traces in A and B. (F) Activation time constants measured from simulated currents in A and B. (G) Steady state current–voltage relationship for simulated currents in C and D.
	Figure 15. Ca2+ concentration determines direction of Cl current in intact cells. Normal oocytes (A and B) or oocytes heterologously expressing iGluR3 (C–E) were bathed in normal Ringer solution (A and B) or NMDG-Ringer (C–E) and were voltage clamped with two microelectrodes, injected with 23 nl 1 mM IP3 (arrows), and exposed to 100 μM kainic acid (KA) in the bath as indicated (bar) (Kuruma and Hartzell 1999). The voltage protocol was an ∼1-s duration pulse to +40 mV, followed by an ∼1-s pulse to −120 mV from a holding potential of −35 mV. (A and C) Plot of current amplitudes during the experiment. (▪) Outward current at the end of the +40 mV pulse, (○ and •) peak inward current at end of the −120-mV pulse. (B and D) Traces a–c corresponding to times indicated in A and C. (E) Ca fluorescence in same oocyte shown in C and D. The oocyte was injected with Ca-green dextran and imaged by confocal microscopy during the +40-mV (▪) and −120-mV (○) voltage-clamp pulses, as previously described (Machaca and Hartzell 1999).

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