J Physiol. 2022 Mar 09.
KEY POINTS: Ionic composition and pH within intracellular compartments, such as endo-lysosomes, rely on the activity of chloride/proton transporters including ClC-6. Distinct CLCN6 mutations previously were found in individuals with neurodegenerative disease, and also putatively associated with neuronal ceroidal lipofuscinosis. Limited knowledge of wild-type ClC-6 transport function impedes understanding of mechanisms underlying these conditions. We resolved transient and transport currents that permit measurement of voltage- and pH- dependences, as well as kinetics, for wild-type and disease-associated mutant ClC-6s. These findings define wild-type ClC-6 function robustly, and reveal how alterations of the slow activation gating of the transporter cause different kinds of neurological diseases.
ABSTRACT: ClC-6 is an intracellularly localized member of the CLC family of chloride transport proteins. It presumably functions in the endo-lysosomal compartment as a chloride-proton antiporter, despite a paucity of biophysical studies in direct support. Observations of lysosomal storage disease, as well as neurodegenerative disorders, emerge with its disruption by knockout or mutation, respectively. An incomplete understanding of wild type ClC-6 function obscures clear mechanistic insight into disease etiology. Here, high-resolution recording protocols that incorporate extreme voltage pulses permit detailed biophysical measurement and analysis of transient capacitive, as well as ionic transport currents. This approach reveals that wild type ClC-6 activation and transport require depolarization to voltages beyond 140 mV. Mutant Y553C associated with early-onset neurodegeneration exerts gain-of-function by shifting the half-maximal voltage for activation to less depolarized voltages. Moreover, we show that the E267A proton glutamate mutant conserves transport currents, albeit reduced. Lastly, the positive shift in activation voltage shown by V580M, a mutant identified in a patient with late- onset lysosomal storage disease, can explain loss-of-function leading to disease. Abstract figure legend CLC transport proteins comprise both channels and transporters. Vesicular CLC transporters function to regulate compartmental ionic homeostasis and acidification. ClC-6 is a vesicular CLC that localizes to the endo-lysosomal compartment. Functional plasma membrane overexpression of GFP-tagged ClC-6 in HEK293 cells surmounted spatial inaccessibility, and rapid whole cell patch recording protocols enabling resolution of fast capacitive transients, as well as ionic transport currents, provided details of wild-type ClC-6 biophysical properties including voltage-dependence, pH-dependence, and kinetics. Clearly defined wild-type ClC-6 function permitted subsequent comparative analysis of mutants, including but not limited to those pertinent to disease. These range from one causing severe, early-onset neurodegeneration, to two variants previously identified in Kufs disease, a late-onset lysosomal storage disease characterized by neuronal ceroid lipofuscinosis. These findings further inform models whereby disruption of ClC-6 biophysical properties set the stage for dysregulated compartmental homeostasis and hence, disease. This article is protected by copyright. All rights reserved.
Keywords: Kuf disease; anion transport; endosome; organellar homeostasis; vesicular CLC