Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid

Abstract

1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

Figure 1

Effect of RS-(±) 2-(4-chlorophenoxy)propionic acid (RS-(±) CPP) on currents through rClC-1. Cl⁻ currents were recorded in response to 100 ms voltage steps ranging from −120 mV to +80 mV (in 20 mV increments) after a 100 ms prepulse to +40 mV from a holding potential of −30 mV. Whole-cell recording showing Cl⁻ currents recorded in (a) standard bath conditions of 178 mM Cl⁻, pH_o 7.4, and (b) with RS-(±) CPP (1 mM) present. Similar results were seen in six cells.

Figure 2

Effect of RS-(±) CPP and its enantiomers on apparent open probability (P_o) of rClC-1. Cl⁻ currents were recorded in response to 100 ms voltage steps between +80 and −140 mV (20 mV increments) from a holding potential of −30 mV, followed by a constant ‘tail' pulse of −100 mV for 50 ms. Apparent P_o was determined from the tail currents by normalizing to the maximal current flowing after the most positive test pulse. (a) Apparent P_o curves in standard bath conditions of 178 mM Cl⁻, pH_o 7.4 (control; n=33) and with RS-(±) CPP (1 mM) present in the bath solution (n=6). The lines represent fits of the Boltzmann distribution (Equation 2). (b) The concentration-dependence of the shift in V_1/2 produced by RS-(±) CPP (n=6), S-(−) CPP (n=5) and R-(+) CPP (n=8). The V_1/2 of channel apparent P_o was determined from the fit of the Boltzmann distribution (Equation 2) at each concentration of drug. Data was fitted with a sigmoidal function of variable slope. Liquid junction potentials have been corrected. Results are expressed as mean±s.e.mean.

Figure 3

Effect of RS-(±) CPP on the binding affinity of the ClC-1 gating site for Cl⁻. The apparent open probability (P_o) at −40 mV was measured as described in Figure 2 in cells in bath solutions of different Cl⁻ concentrations in the absence (control) and presence of 1 mM RS-(±) CPP added to the bath. The curves represent the fit of equation 3. Results are expressed as mean±s.e.mean (n=3–6).

Figure 4

Effect of RS-(±) 2-(4-chlorophenoxy)propionic acid (RS-(±) CPP) on currents through rClC-1 at a +20 mV holding potential. The voltage protocol applied is the same as that described in Figure 1, however from a holding potential of +20 mV. Whole-cell Cl⁻ currents were recorded in (a) standard bath conditions of 178 mM Cl⁻, pH_o 7.4, and (b) with RS-(±) CPP (1 mM) present. Similar results were seen in four cells. (c) Effect of RS-(±) CPP (1 mM) on instantaneous (I_max: equation 1, t=0) and steady state (C: equation 1, t=∞) current-voltage relationships at a +20 mV holding potential. All values for each cell are normalized to the peak instantaneous current at −120 mV in standard bath conditions of 178 mM Cl⁻, pH_o 7.4, without RS-(±) CPP present. Results are expressed as mean±s.e.mean (n=4).

Figure 5

Concentration-dependent reduction of steady state current through rClC-1 produced by RS-(±) CPP and its enantiomers (i.e. S-(−)- and R-(+) CPP). Currents were recorded in response to the voltage protocol described in Figure 1. Analysis of currents was performed on values for C, the steady state component, derived from equation 1 at −80 mV at different concentrations of each compound. The degree of reduction of the Cl⁻ current for each cell is measured as a fraction of the current in the absence of the compound (I/I_control). Curves were fitted with a sigmoidal function of variable slope. Results are expressed as mean±s.e.mean (n=5–8)

Figure 6

Effect of RS-(±) CPP on currents through rClC-1 at low external pH (pH_o 6.0). The voltage protocol applied is the same as that described in Figure 1. Whole-cell Cl⁻ currents were recorded in (a) a bath solution of 178 mM Cl⁻, pH_o 6.0, or (b) with RS-(±) CPP (1 mM) present in the bath solution at pH_o 6.0. Similar results were seen in five cells.