Electro-pharmacological profiles of two brain mitoplast anion channels: Inferences from single channel recording

Abstract

We have characterized the conduction and blocking properties of two different chloride channels from brain mitochondrial inner membranes after incorporation into planar lipid bilayers. Our experiments revealed the existence of channels with a mean conductance of 158 ± 7 and 301 ± 8 pS in asymmetrical 200 mM cis/50 mM trans KCl solutions. We determined that the channels were ten times more permeable for Cl^- than for K⁺, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channels were bell-shaped voltage dependent, with maximum open probability 0.9 at ± 20 mV. Two mitochondrial chloride channels were blocked after the addition of 10 µM DIDS. In addition, 158 pS chloride channel was blocked by 300 nM NPPB, acidic pH and 2.5 mM ATP, whereas the 301 pS chloride channel was blocked by 600 µM NPPB but not by acidic pH or ATP. Gating and conducting behaviors of these channels were unaffected by Ca²⁺. These results demonstrate that the 158 pS anion channel present in brain mitochondrial inner membrane, is probably identical to IMAC and 301 pS Cl channel displays different properties than those classically described for mitochondrial anion channels.

Keywords: brain; chloride channels; intracellular ion channels; mitochondria; mitoplast; single channel.

Figure 1. Single channel recordings, current/voltage curve and channel open probability as a function of voltages. (A and B) Single channel recordings in 200/50 mM KCl (cis/trans) at ±50 mV (the arrow indicates the closed state). (C) Current-volt age characteristics of single channel events in a 200/50 mM KCl (cis/trans) gradient solution, 301 pS channel (□) and 158 pS Channel (■). Error bars indicate the S.E. from 8 independent experiments. (D and E) Channel open probability (Po)-voltage curves.

Figure 2. The effect of DIDS on channels gating behaviors at positive and negative voltages. (A and B) Single-channel recordings under control conditions (200/50 mM KCl; cis/trans) and immediately after cis addition of DIDS 10 µM (n = 4). Arrows indicate the closed levels.

Figure 3. The effect of NPPB on channel activity at different voltages. (A and B) Representative recordings of channel currents under control conditions and after addition of 300 and 600 µM NPPB to cis face. Channels' activities are completely blocked at negative and positive potentials (n = 4). Arrows indicate the closed levels.

Figure 4. The effect of ATP on channels' gating behaviors. (A and B) Single channels' activities in control conditions and after adding 2.5 mM ATP to cis face (n = 4). The arrows indicate the closed levels.

Figure 5. The effect of pH on anion channel activity. (A and B) Single channel recordings under control conditions (200/50 mM KCl; cis/trans, pH 7.2) and cis acidic pH (pH 5.8). Significant differences in the open probability value and amplitude of 158 pS anion channel are observed (n = 4). Arrows indicate the closed levels.

Figure 6. The effect of Ca²⁺ ions on channel behavior. (A and B) Single-channel activities in control condition (10 µM calcium ions) and after addition of 1 mM EGTA (cis) in the same bilayer. Arrows indicate closed state of the channel.