Cytoplasmic ATP inhibition of CLC-1 is enhanced by low pH

Abstract

The CLC-1 Cl(-) channel is abundantly expressed on the plasma membrane of muscle cells, and the membrane potential of muscle cells is largely controlled by the activity of this Cl(-) channel. Previous studies showed that low intracellular pH increases the overall open probability of recombinant CLC-1 channels in various expression systems. Low intracellular pH, however, is known to inhibit the Cl(-) conductance on the native muscle membrane, contradicting the findings from the recombinant CLC-1 channels in expressed systems. Here we show that in the presence of physiological concentrations of ATP, reduction of the intracellular pH indeed inhibits the expressed CLC-1, mostly by decreasing the open probability of the common gate of the channel.

Figure 1.

Effects of 1 mM cytoplasmic ATP on CLC-1 at three intracellular pH conditions. (A) Recording traces were obtained in the indicated pH and ATP conditions using voltage protocol A. (B) Normalized current (I_norm) represents the initial, tail current normalized to the maximal initial current in the absence of ATP. Each data point is the average from 3–6 patches. This I_norm value reflects the product of the fast-gate P_o and the common-gate P_o, namely P_o ^f × P_o ^c. Solid and open symbols were in 0 and 1 mM cytoplasmic ATP, respectively.

Figure 2.

Effects of 1 mM ATP on the common gate of CLC-1. (A) Recording traces obtained in the indicated conditions using voltage protocol B. (B) Normalized value of the initial tail current (I_norm) in each pH condition (as shown in A). This I_norm value has been widely used to represent the P_o of the common gate (P_o ^c). Dividing the I_norm in Fig. 1 B (from protocol A) by the I_norm here (from protocol B) gives the fast-gate P_o (P_o ^f), which is shown in the inset of each panel. Solid and open symbols were obtained in 0 and 1 mM cytoplasmic ATP, respectively.

Figure 3.

Dependence of the V_1/2 of the common-gate P_o ^c–V curve on the ATP concentration in three different pH conditions. Each data point is the average from 3–7 patches. Solid curves are drawn according to a Michaelis-Menten equation with the ATP half-effective concentration and the saturated V_1/2 value of 0.31 mM and −20 mV (pH 7.4), 0.40 mM and +49 mV (pH 6.8), and 0.12 mM and +91 mV (pH 6.2).

Figure 4.

Reversible ATP inhibition on the CLC-1 common-gate activity at pH 6.8. Top panels show recording traces by a pulse protocol of +40 mV test voltage (300 ms), followed by the short pulse to +170 mV (400 μs), and finally the tail voltage step at −120 mV. The recorded traces were shown around the initial tail current for those traces during ATP wash-in (left) and wash-out (right). Each recording trace is separated by 2 s, and the initial value of the tail current in each trace is plotted against time at the bottom panel. The red curve represents a single-exponential fit with a time constant of 3.4 s, which does not fit the ATP wash-in process well. Three other patches show the same results from such ATP wash-in and wash-out experiments.