Can ClC-2 Chloride Channel be Activated by Hyperpolarization Alone in Cells Dialyzed with Non-Permeant Anions?

Abstract

Open and closing of ion channels is driven by conformational changes triggered by either an intrinsic voltage sensor in voltage-gated channels or by ligand binding in ligand-gated channels. However, ClC-2, a two pore Cl− channel is activated by hyperpolarisations despite of lacking of an intrinsic voltage sensor. The structural information available to date strongly suggests that in the closed conformation each pore of ClC-2 is occluded by the negatively charged side chain of a glutamate residue. Although the gating mechanism is unknown, there is evidence which indicates that intracellular anions and extracellular protons regulate gating. The present work was aimed to determine the contribution of pore occupancy caused by intracellular anions in gating the mouse ClC-2 channel. In our experiments, ClC-2 was readily activated by hyperpolarisations when permeant anions such Cl−, SCN−, Br− and I− were present on either side of the membrane. However, ClC-2 was not activated in cells dialyzed with acetate (0 Cl−) and exposed to [H+]O=10−7.3 M. In contrast, the channels were opened by increasing [Cl−]i at [H+]O=10−10 M_a condition unlikely to protonate the glutamate's side chain. Importantly, voltage gating occurred when F− or glutamate were present in the cytosolic side. Since these two anions are non-permeant we propose that they must enter the pore and interact with the glutamate side chain from the inside in order to induce opening. Thus, we propose that a strong hyperpolarisation drives the intracellular anions into the permeation pathway and the subsequent electrostatic interaction between the anions and the negatively charged side chain opens the pore. Our data support the hypothesis that voltage-dependent gating in mouse ClC-2 requires intracellular anions. Supported by grant 219949 from CONACyT.