ClC-K chloride channels are crucial for sodium chloride reabsorption in the loop of Henle and for potassium secretion in the inner ear. Human ClC-K channels require the accessory subunit barttin for surface membrane insertion and to be active. The rodent homolog rClC-K1 is functional also in the absence of barttin, and barttin only modifies its voltage dependence.

To define regions within barttin that are important for associating to ClC-K or to exert the distinct functional effects, we individually substituted every amino acid by tryptophan and studied the effects of these mutations on subcellular distribution as well as on the function of hClC-Ka or rClC-K1.

Only four, I9W, F11W, L16W, L19W, out of 18 point mutations in TM1, but 14 of 20 TM2 mutations leave the chaperone function and the functional effects of barttin unaffected. There are only two substitutions, F24W and M26W, that completely abolish colocalization and functional effects on rClC-K1 gating. Several mutations within TM1 and TM2 affect trafficking of barttin, resulting in intracellular retention of pore-forming and accessory subunit. These mutant barttins reduce rClC-K1 currents below values in the absence of barttin. Other TM1 mutations leave trafficking unaffected, but modify the gating effect of barttin. We conclude that the interaction between barttin and ClC-K is determined by multiple barttin side chains. Moreover, residues within TM1 as well as within TM2 determine the chaperone function of barttin. Lastly, residues within TM1 define the gating modification of rClC-K1. Our experiments illustrate the functional complexity of barttin.