Intracellular chloride channels (CLICs) are implicated in physiological processes, and they exist in membranous and soluble forms. CLIC3 is present in lysosomes, where it is involved in the regulation of the recycling pathway. While pH is known to regulate channel activity of CLICs, the precise mechanism for CLIC3 is not yet elucidated. Here, we report the pH regulation of transmembrane CLIC3 for the first time and identify a key residue mediating this intrinsic property. CLIC3 was ectopically expressed in HEK-293 cells, where it readily localized to the plasma membrane. pH-dependent activation of CLIC3 was assessed using on-cell, whole-cell, and inside-out patch-clamp recordings. The pH was adjusted by adding HCl and CsOH (respectively) to the bath solution. The current density increased (by 48.5%) when the pH in the bath solution was changed from 7.2 to 5.2. Here, through comprehensive mutagenesis and electrophysiological studies, we identified a critical histidine residue (H21) in the N terminus and (H181) in the C terminus as potential pH sensors for CLIC3. The H21A and H181A mutations abolished the changes in current density when pH was changed from 7.2 to 5.2. In summary, we deciphered the mechanism involving H21 and H181 in the regulation of conductance and pH-dependent activation of transmembrane CLIC3.
Loyo-Celis et al. (Sun,) studied this question.