8.A.53 The CLC-K Accessory Subunit, Barttin, (Barttin) Family
CLC-K chloride channels (TC# 2.A.49.2.4-5) are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion (Scholl et al. 2006). Human ClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. Scholl et al. 2006 mapped the separate functions of barttin to structural domains by deletion analyses. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin may be necessary for tight adjustment of epithelial Cl- conductances to ensure precise regulation of body salt content and endocochlear potential.
As noted above, Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction and gating. Wojciechowski et al. 2015 performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two N-terminal transmembrane helices followed by a long intracellular carboxy-terminus, and the transmembrane core of barttin suffices for most effects on the α-subunit. After substitution of every amino acyl residue in the two predicted TMSs (aa9-26 and 35-55) with tryptophan, the co-expressed mutant barttin with wild type human ClC-Ka had no effect on endoplasmic reticulum exit and surface membrane insertion. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrated that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect the barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking (Wojciechowski et al. 2015).