The impact of patient mutations in Na/phosphate cotransporters to function and trafficking of the protein (360G-Wellcome-211750_Z_18_Z)

The renal transport system for inorganic phosphate (Pi), SLC34A1, is central to balancing Pi levels in the human body. Mutations in SLC34A1 have been identified in patients with isolated renal phosphate wasting, generalised proximal tubulopathies (renal Fanconi’s syndrome), renal stone formers and patients with nephrocalcinosis. The spectrum of phenotypes observed points to a complex interplay between the nature of the mutation and the genetic background of the patient. Intriguingly, some of the mutations show a dominant phenotype. The aim of this proposal is to investigate the molecular and cellular determinants for the dominant behaviour of certain SLC34A1 mutations. We have identified two patients with mutations in SLC34A1. The first patient carries a heterozygous SLC34A1 mutation Ile456Gln whilst the second is compound heterozygous for Arg512Cys and a deletion of Val91-Ala97. These mutations will be used to perform functional analyses in Xenopus oocytes and epithelial cell lines co-expressing wild-type and mutated transporters. Chimeric wt-mutant constructs will also be generated to exclude differences in intracellular processing of the individual units. Intracellular trafficking will be tested in renal epithelial cell lines using wild-type and mutated transporters with different fluorescent tags followed by confocal microscopy.