Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor.
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.
Traverso
Sonia
S
Istituto di Biofisica, Sezione di Genova, CNR, via de Marini, 6, I-16149 Genova, Italy.
Elia
Laura
L
Pusch
Michael
M
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
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