Behind the Scenes of CLC Gating: Deriving the Voltage Dependence of Membrane Proteins by Admittance Measurements

CLC channels and transporters are expressed in virtually every living cell and fulfill a number of housekeeping functions such as stabilizing the resting potential of skeletal muscles, controlling renal salt excretion, and regulating [Cl−] and pH in diverse cell organelles (1). The physiological importance of the CLC family is emphasized by the existence of various human diseases associated with mutations in genes encoding CLC channels or transporters. The CLC family encompasses anion channels and secondary-active Cl−/H+ exchangers (2), and thus contains proteins that are mediators of thermodynamically different transport processes. Despite these principal differences in function, all known CLC isoforms seem to be regulated by complex voltage-dependent gating. The importance of this regulation is illustrated by a large number of disease-associated mutations that specifically modify the voltage-dependence of various members of the CLC family.Gating of CLC channels and transporters are both related to conformational changes that underlie the coupled exchange cycle in the isoforms belonging to the transporter branch. High-resolution, three-dimensional structures of pro- and eukaryotic CLC transporters in different conformations provide insights into the structural rearrangement underlying coupled transport as well as the channel and transporter gating. This structural information revealed the existence of a highly conserved glutamate side chain at the extracellular side that can either project into the aqueous external solution or occupy two of three anion-binding sites in the CLC anion selectivity filter (3 and 4). This glutamate side chain—often referred to as “gating glutamate”—is apparently moving in two sequential steps from the outside into the anion conduction pathway of the protein (Fig. 1A). Surprisingly, there are no functional data that demonstrate the existence of such sequential steps in mammalian CLC proteins. Gating of CLC transporters is perfectly well described with a simple two-state gating scheme. The nonlinear capacitances associated with activation gating are also monophasic and change upon voltage steps with monoexponential time dependences. This inconsistency is now addressed in an exciting new study by Grieschat and Alekov in this issue of the Biophysical Journal ( 5), and it is used to provide novel insights into the mechanisms underlying CLC channel/transporter gating