UNLABELLED: Mutations in the gene encoding the human sodium-phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant-negative behavior, were described. We found no evidence of altered kinetics or dominant-negative effects. Thus, the mutations cannot account for the clinical phenotype. INTRODUCTION: Mutations in NPT22a, the gene encoding the sodium-phosphate cotransporter NaPi-IIa, were for the first time linked to human disease by Priè and colleagues. Two patients are described with renal phosphate wasting who were heterozygous for either the A48F or V147M mutation. Expressed in Xenopus oocytes, both mutants showed reduced phosphate affinity. Furthermore, coexpression of mutants with wildtype (WT) NaPi-IIa resulted in reduced cotransport function, explaining the mutants' dominant-negative effect in the patients. Intrigued by the implications of these findings on transporter kinetics, we decided to examine the transport characteristics of the two mutants in more detail. MATERIALS AND METHODS: We recreated the two mutants, expressed them in Xenopus oocytes, and analyzed their kinetic behavior by two-electrode voltage clamp. We also performed coexpression experiments where we injected mRNA for WT and mutants containing an additional S462C mutation, enabling complete inhibition of cotransport function with cysteine-modifying reagents. Finally, we expressed WT and mutant NaPi-IIa as C-terminal fusions to green fluorescent protein (GFP) in opossum kidney (OK) cells. RESULTS AND CONCLUSIONS: We found in our oocyte expression experiments that P(i)-induced currents were reduced in both mutants, whereas P(i) and Na affinities and other transport characteristics were not affected. The amount of cotransport activity remaining after cysteine modification, corresponding to WT activity, was not affected by coexpression of either mutant. Finally, GFP-tagged WT and mutants were expressed at the apical membrane in OK cells, showing that both mutants are correctly targeted in a mammalian cell. In conclusion, our data from oocyte and OK cell expression studies suggest that the heterozygous A48F and V 147M mutations cannot explain the pathological phenotype observed by Priè and colleagues.
UNLABELLED: Mutations in the gene encoding the humansodium-phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant-negative behavior, were described. We found no evidence of altered kinetics or dominant-negative effects. Thus, the mutations cannot account for the clinical phenotype. INTRODUCTION: Mutations in NPT22a, the gene encoding the sodium-phosphate cotransporterNaPi-IIa, were for the first time linked to human disease by Priè and colleagues. Two patients are described with renal phosphate wasting who were heterozygous for either the A48F or V147M mutation. Expressed in Xenopus oocytes, both mutants showed reduced phosphate affinity. Furthermore, coexpression of mutants with wildtype (WT) NaPi-IIa resulted in reduced cotransport function, explaining the mutants' dominant-negative effect in the patients. Intrigued by the implications of these findings on transporter kinetics, we decided to examine the transport characteristics of the two mutants in more detail. MATERIALS AND METHODS: We recreated the two mutants, expressed them in Xenopus oocytes, and analyzed their kinetic behavior by two-electrode voltage clamp. We also performed coexpression experiments where we injected mRNA for WT and mutants containing an additional S462C mutation, enabling complete inhibition of cotransport function with cysteine-modifying reagents. Finally, we expressed WT and mutant NaPi-IIa as C-terminal fusions to green fluorescent protein (GFP) in opossum kidney (OK) cells. RESULTS AND CONCLUSIONS: We found in our oocyte expression experiments that P(i)-induced currents were reduced in both mutants, whereas P(i) and Na affinities and other transport characteristics were not affected. The amount of cotransport activity remaining after cysteine modification, corresponding to WT activity, was not affected by coexpression of either mutant. Finally, GFP-tagged WT and mutants were expressed at the apical membrane in OK cells, showing that both mutants are correctly targeted in a mammalian cell. In conclusion, our data from oocyte and OK cell expression studies suggest that the heterozygous A48F and V 147M mutations cannot explain the pathological phenotype observed by Priè and colleagues.
Authors: Karl P Schlingmann; Justyna Ruminska; Martin Kaufmann; Ismail Dursun; Monica Patti; Birgitta Kranz; Ewa Pronicka; Elzbieta Ciara; Teoman Akcay; Derya Bulus; Elisabeth A M Cornelissen; Aneta Gawlik; Przemysław Sikora; Ludwig Patzer; Matthias Galiano; Veselin Boyadzhiev; Miroslav Dumic; Asaf Vivante; Robert Kleta; Benjamin Dekel; Elena Levtchenko; René J Bindels; Stephan Rust; Ian C Forster; Nati Hernando; Glenville Jones; Carsten A Wagner; Martin Konrad Journal: J Am Soc Nephrol Date: 2015-06-05 Impact factor: 10.121