title: 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 approximately 4 micro M). 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.
authors.affiliation: Istituto di Biofisica, Sezione di Genova, CNR, via de Marini, 6, I-16149 Genova, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Traverso
authors.forename: Laura
authors.initials: L
authors.lastname: Elia
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000409:Alanine / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006706:Homeostasis; D015640:Ion Channel Gating; D007700:Kinetics; D016014:Linear Models; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D012694:Serine / Q000235:genetics; D014101:Torpedo; D014981:Xenopus
chemical_list: C120501:CLC-0 chloride channel protein, Torpedo californica; D018118:Chloride Channels; D015084:2,4-Dichlorophenoxyacetic Acid; D018698:Glutamic Acid; D012694:Serine; C007017:4-chlorophenoxyacetic acid; D000409:Alanine