INTRODUCTION: Mutations in KCNJ10 lead to epilepsia, ataxia, sensorineural hearing loss and a tubulopathy, with hypomagnesemia and mild salt loss reminiscent of Gitelman’s syndrome. All mutations studied so far affect channel activity and/or trafficking. To study the effect of KCNJ10 loss of function in brain, we generated a zebrafish model of the disease OBJECTIVES: KCNJ10 mutations were expressed in mammalian cells and oocytes to investigate the properties of mutant channels in more detail. Previous data had suggested that some mutations are partially functional and we hypothesized – based on structural considerations, that these mutations impinge on channel regulation. Animal models were studied to elucidate physiological aspects of KCNJ10 function. METHODS: KCNJ10 WT and R65P, G77R and R175Q were expressed in CHO cells and oocytes and assessed using whole cell patch-clamp, single channel recordings and macropatch recordings. Epileptic activity was recorded in morpholino knock-down zebrafish using a new recording technique. RESULTS: All mutations investigated strongly reduced the open probability of KCNJ10 and KCNJ10/KCNJ16 heteromeric channels. R65P led to a reduction in mean channel open time, and all mutations shifted the pH-dependence of activation to more alkaline pH values. R175Q showed a strong reduction in PIP2 affinity. Zebrafish injected with MO against KCNJ10 showed rhythmic abnormal motor patterns and hyperactivity in the optic tectum, resembling activity observed after treatment with a known seizure-inducing agent. DISCUSSION: These results provide a framework to understand, and possibly modify, loss of function in patients with mutations in KCNJ10. In the kidney, KCNJ10 is critically involved in salt reabsorption via NCC, both by providing pump-leak coupling for the Na+/K+-ATPase as well as by providing the driving force for chloride exit through ClC-K/barttin channels. KCNJ10 is critical for stabilizing the neuronal membrane voltage by allowing glial cells to remove K+ from sites of high neuronal activity, and it is critical for the generation of the endocochlear potential. MO knockdown zebrafish are a valuable model to study the neuronal phenotype of KCNJ10 deficiency.