Inward rectification in ClC-0 chloride channels caused by mutations in several protein regions

Abstract

Several cloned ClC-type Cl- channels open and close in a voltage-dependent manner. The Torpedo electric organ Cl- channel, ClC-0, is the best studied member of this gene family. ClC-0 is gated by a fast and a slow gating mechanism of opposite voltage direction. Fast gating is dependent on voltage and on the external and internal Cl- concentration, and it has been proposed that the permeant anion serves as the gating charge in ClC-0 (Pusch, M., U. Ludewig, A. Rehfeldt, and T.J. Jentsch. 1995. Nature (Lond.). 373:527-531). The deactivation at negative voltages of the muscular ClC-1 channel is similar but not identical to ClC-0. Different from the extrinsic voltage dependence suggested for ClC-0, an intrinsic voltage sensor had been proposed to underlie the voltage dependence in ClC-1 (Fahlke, C., R. Rüdel, N. Mitrovic, M. Zhou, and A.L. George. 1995. Neuron. 15:463-472; Fahlke, C., A. Rosenbohm, N. Mitrovic, A.L. George, and R. Rüdel. 1996. Biophys. J. 71:695-706). The gating model for ClC-1 was partially based on the properties of a point-mutation found in recessice myotonia (D136G). Here we investigate the functional effects of mutating the corresponding residue in ClC-0 (D70). Both the corresponding charge neutralization (D70G) and a charge conserving mutation (D70E) led to an inwardly rectifying phenotype resembling that of ClC-1 (D136G). Several other mutations at very different positions in ClC-0 (K165R, H472K, S475T, E482D, T484S, T484Q), however, also led to a similar phenotype. In one of these mutants (T484S) the typical wild-type gating, characterized by a deactivation at negative voltages, can be partially restored by using external perchlorate (ClO4-) solutions. We conclude that gating in ClC-0 and ClC-1 is due to similar mechanisms. The negative charge at position 70 in ClC-0 does not specifically confer the voltage sensitivity in ClC-channels, and there is no need to postulate an intrinsic voltage sensor in ClC-channels.

Figure 1

Effect of mutant D70G on gating of ClC-0. (A) Voltage protocol for all traces shown in this paper. (B) Macroscopic currents from ClC-0 WT deactivate after a pulse to +40 mV. (C) Macroscopic currents from mutant D70G. Channels open at negative potentials after a step from a positive pre-potential.

Figure 2

Several point mutations convert ClC-0 into inwardly rectifying channels. Macroscopic currents from 2-electrode voltage clamp experiments are shown for mutants D70E (A), K165R (B), H472K (C), S475T (D), E482D (E), and T484Q (F).

Figure 5

Analysis of effects of external ClO₄ ⁻ on WT and mutant T484S channels. (A) Macroscopic 2-electrode-voltage-clamp data from WT in 100 mM Cl⁻ (left), a mixture of 90 mM Cl⁻ and 10 mM ClO₄ ⁻ (center), and 100 mM ClO₄ ⁻ (right). ClO₄ ⁻ blocks ClC-0 in a voltage dependent manner with the following parameters: δ = 0.30 ± 0.02; K _d(0 mV) = 2.2 ± 0.5 mM (n = 3). (B) Stimulation of gating in T484S mutant channels by ClO₄ ⁻. Macroscopic currents from T484S in 100 mM Cl⁻ (left), a mixture of 90 mM Cl⁻ and 10 mM ClO₄ ⁻ (center), and 100 mM ClO₄ ⁻ (right).

Figure 3

(A) Location of mutations that lead to inwardly rectifying channels. They are located in domain 1 (D70), at the carboxy-terminal end of domain 3 (K165), in domain 11 (H472, S475) and between domain 11 and domain 12 (E482, T484). (B) Alignment of homologous ClC proteins in the region of the domains D11/D12. hClC-1 is the human muscular chloride channel (Steinmeyer et al., 1991; Koch et al., 1992), ClC-2 is a ubiquitous swelling-activated Cl⁻ channel from rat (Thiemann et al., 1992), ClC-5 is predominantly expressed in kidney (Steinmeyer et al., 1995; Lloyd et al., 1996), ClC-7 is a rather broadly expressed CLC-protein (Brandt and Jentsch, 1995), and scClC is the S. cerevisiae CLC protein GEF-1 (Greene et al., 1993). The prefix r in front of ClC means rat, h means human, and sc means yeast.

Figure 4

Macroscopic currents from T484S mutant channels in an inside-out patch clamp experiment.