PHYSIOLOGY / BASIC

Introduction and Aims:The recent improvements of next generation sequencing techniques allow relatively rapid and cheap identification of new gene mutations in patients with familial hypomagnesemia.Over the last years, many new genes are identified that regulate Mg2+ reabsorption in the kidney.However, translating the genetic findings into functional assays to examine the function of the affected genes remains challenging because of inadequate cell models, the absence of an Mg2+ radioisotope and limited availability of animal models.Methods: To elucidate the physiological role of newly identified magnesium transporters, a new Mg2+ transport assay using the stable 25Mg isotope was established.Moreover, the zebrafish knockdown model is used to study the in vivo functions.Results: For example, we have identified new mutations in the gene CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia.For the first time, a recessive mode of inheritance of CNNM2 mutations was observed and mutations in CNNM2 are associated with mental disability.Using stable Mg2+ isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg2+-permeable cation channel TRPM7.In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg2+ uptake.Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development and reduced body Mg content.These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes.Altogether these data show that CNNM2 is fundamental for brain development, neurological functioning and Mg2+ homeostasis.Conclusions: By establishing a novel Mg2+ transport assay using stable Mg2+ isotopes and the loss-of-function zebrafish model, we provide a unique system to examine the function of novel genes in Mg2+ homeostasis.These new in vitro and in vivo models may aid to explain the function of electrolyte transporters in the future.