Bartter´s syndrome type IV is an inherited syndrome characterized by renal salt wasting and sensorineural deafness. It is caused by mutations in barttin, an accessory subunit of renal and inner ear ClC-K chloride channels that promotes channel insertion into the surface membrane and switches hClC-Kb into an anion-conducting state. We here employed heterologous expression in mammalian cells and patch-clamping to delineate the functional consequences of various disease-causing mutations. Several mutant barttins were reported not to induce anion currents in cells expressing hClC-Kb. To differentiate effects on hClC-Kb trafficking and function, we studied the effect of mutant barttins using a hClC-1-hClC-Kb concatamer that inserts into the surface membrane independently of barttin. hClC-Kb was inactive in these heterodimers when co-expressed with three mutations (R8L, R8W, and G10S). G47R, found in patients with less severe symptoms, partially activated hClC-Kb, indicating that this particular function is a determinant of the disease severity. R8L, R8W, G10S and G47R caused characteristic gating alterations in another ClC-K isoform, rClC-K1, demonstrating that mutant barttins are capable to associate with ClC-K channels. Different functions of barttin thus appear to be mediated by distinct domains of the protein. We conclude that a major effect of disease-causing barttin mutations is the failure to switch hClC-Kb into an anion-conducting state.