Conformational changes required for H+/Cl− exchange mediated by a CLC transporter

Abstract

CLC-type exchangers mediate transmembrane Cl– transport. Mutations altering their gating properties cause numerous genetic disorders. However, their transport mechanism remains poorly understood. In conventional models two gates alternatively expose substrate(s) to the intra- or extra-cellular solutions. In the CLCs, a glutamate was identified as the only gate; suggesting that they function according to a non-conventional mechanism. Here we show that transport in CLC-ec1, a prokaryotic homologue, is inhibited by crosslinks constraining movement of helix O far from the transport pathway. Crosslinked CLC-ec1 adopts a wild type-like structure, indicating stabilization of a native conformation. Movements of helix O are transduced to the ion pathway via a direct contact between its C-terminus and a tyrosine, a constitutive element of the second gate of CLC transporters. Therefore, the CLC exchangers have two gates that are coupled through conformational rearrangements outside the ion pathway.

Shared Molecular Mechanisms of Membrane Transporters

“…Interestingly, chlorideproton exchangers (ClCs) (6) were thought, until recently, to be an exception to this rule because transport seemed to be associated only with the local movements of side chains within a rigid protein scaffold (7)(8)(9). However, new data suggest that global structural transitions are also likely to be part of the ClC mechanism (10)(11)(12). It should be noted that small perturbations, such as mutations or the binding of small molecules, can turn some transporters into channels, presumably by stabilizing the states in which both the extracellular and intracellular gates are open (e.g., a double mutant of the Escherichia coli ClC transporter becomes a channel) (6,13,14).…”

ClC-5: Physiological role and biophysical mechanisms

“…This mechanism has been challenged by Accardi and colleagues who proposed that the coupling mechanism rather than being of kinetic nature, is a consequence of the thermodynamic stoichiometry of substrate binding measured at equilibrium by isothermal titration calorimetry (ITC) [74]. It has been also suggested [90] that transport coupling involves an intracellular gate formed by the conserved tyrosine residue Y445 (ClC-ec1 numbering) [75,80].…”

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl^- Channels and Transporters

“…However, the fraction of liposomes containing at least one active protein, f 0 , can still be determined by adding detergent and measuring the residual trapped Cl content (step 6.8). This allows for the determination of the molecular mass of the protein by carrying out a protein titration and using Equation 10.…”