Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

Abstract

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

A Two-Holed Story: Structural Secrets About ClC Proteins Become Unraveled?

“…All three mutations studied (E166A, E166V, E166Q) almost completely abolished the closure of the protopore gate, resulting in permanently open channels (10) Together, these results have led to the proposal that the gating of ClC channels is regulated by a simple mechanism in which the glutamate side chain acts as a gate occluding the ion pathway. Although several details may not be easily explained (3,47), this simple model is qualitatively in accordance with several properties of ClC-0 gating. First, it is consistent with the independence of the two pores because each pore has its own glutamate.…”

“…However, their gating is strongly dependent on pH, indicating a kind of memory of the bacterial transporter. This notion is also supported by the conserved crucial role of E148 for ClC gating in all ClC channels studied (10,13,14,16,32,40,47). The new findings could be also helpful in future studies on some human channels, such as ClC-6 and ClC-7, that are still poorly understood.…”

Molecular determinants of common gating of a ClC chloride channel

“…The structural basis of protopore gating, inferred from prokaryotic Cl À /H þ exchanger crystal structures 8 , involves an external gate formed by E ext that opens when protonated 19 to reveal S ext 8 . More widespread conformational changes may accompany protopore gating 20 ; however, their nature is unclear.…”

“…First, common gating of ClC-0 25,26 and ClC-1 17 depends on Cl À concentration, hinting at the involvement of residues in the channel pore. Second, neutralization of E ext in ClC channels removes both protopore and common gating, resulting in a constitutively open phenotype 8,19,20,[27][28][29] , whereas, in ClC-1 at least, the wider conformational rearrangements consistent with common gating appear to remain intact 23 . Third, mutation of ClC-0 E ext to aspartate (E166D), which mainly differs from glutamate by having a shorter side chain, greatly reduces open probability of the protopore gate while simultaneously locking the common gate open 19 .…”

Unique gating properties ofC. elegansClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker

“…6,[8][9][10][11] All eukaryotic and some bacterial ClC proteins have extensive cytoplasmic C-terminal domains following the R-helix. [1][2][3][4] These intracellular C-termini contain two cystathionine-β-synthase (CBS) domains.…”