Functional characterization of ClC-K/barttin complexes of different stoichiometries

Abstract

ClC-K chloride channels contribute to salt reabsorption in the kidney and to potassium secretion into the endolymph in the inner ear. They build homodimers with each α-subunit possessing its own conducting protopore. Each protopore is regulated by an individual fast gate, while a common slow gate opens and closes both protopores together. Barttin is the accessory β-subunit of ClC-K channels and affects their expression, subcellular localization and gating properties. Co-immunoprecipitations indicate binding of barttin to B- and J-helices of ClC-K α-subunits assuming a 1:2 α:β-subunit stoichiometry for one protopore. However, functional consequences of varying stoichiometry between both subunits are still unknown. To investigate effects of different subunit stoichiometries on channel function, barttin molecules were covalently linked to hClC-Ka or rClC-K1 in a 1:1, 1:2 and 2:1 α:β-subunit ratio. These concatamers were expressed in HEK293T and MDCKII cells and the impact of these compositions on expression, trafficking and channel gating was analyzed using biochemical methods, confocal microscopy and patch clamp technique. We found that one β- per one α-subunit is sufficient for i) proper transport of the channel to the plasma membrane, ii) hClC-Ka channel activation and iii) constitutive slow gate opening of hClC-Ka and rClC-K1. Two β-subunits per one α-subunit additionally open the fast gate of both orthologous channels constitutively. Therefore, a 1:2 α:β-subunit stoichiometry per protopore allows a complete, functional activation of ClC-K channels. Furthermore, palmitoylation-deficient barttin mutants were incorporated in concatamers with different α:β-subunit ratios to study the impact of barttin palmitoylation on channel properties. We here confirm that barttin palmitoylation is not necessary for channel trafficking but for hClC-Ka activation. Co-expression of WT barttin opens the channel, indicating a dynamic exchange of barttin molecules at their binding sites. Palmitoylation is not necessary for barttin to open the rClC-K1 slow gate constitutively but it is essential for constitutive opening of the rClC-K1 fast gate. In further approaches such concatamers might be used to elucidate functional stoichiometries and consequences of disease causing mutations not only for ClC-K/barttin complexes but also for other protein interaction partners.