Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia.

Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH)2D3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.

Correlating charge movements with local conformational changes of a Na(+)-coupled cotransporter.

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na(+)-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na(+), providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.