scholarly journals A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc

2008 ◽  
Vol 295 (2) ◽  
pp. F371-F379 ◽  
Author(s):  
Graciana Jaureguiberry ◽  
Thomas O. Carpenter ◽  
Stuart Forman ◽  
Harald Jüppner ◽  
Clemens Bergwitz
Keyword(s):  
Sodium Phosphate ◽  
P Uptake ◽  
Complete Loss ◽  
Hypophosphatemic Rickets ◽  
Paternal Allele ◽  
Wild Type ◽  
Ok Cells ◽  
Phosphate Cotransport ◽  
And Function ◽  
Sodium Phosphate Cotransport

The present study describes two novel compound heterozygous mutations, c.410C>T(p.T137M) (T137M) on the maternal and g.4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276–280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-Pi cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[Stop446]hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent 33P uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137]hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g.4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of 22Na and 33P uptake was increased to 7.1 ± 3.65 for EGFP-[M137]hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.

Na+/H+ Exchanger-Regulatory Factor 1 Mediates Inhibition of Phosphate Transport by Parathyroid Hormone and Second Messengers by Acting at Multiple Sites in Opossum Kidney Cells

2003 ◽  
Vol 17 (11) ◽  
pp. 2355-2364 ◽  
Author(s):  
Matthew J. Mahon ◽  
Judith A. Cole ◽  
Eleanor D. Lederer ◽  
Gino V. Segre
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylyl Cyclase ◽  
Sodium Phosphate ◽  
Regulatory Factor ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Kinase C ◽  
Phosphate Cotransport ◽  
Sodium Phosphate Cotransport

Abstract The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH (∼60%) and the inhibition mediated by phorbol 12-myristate 13-acetate (∼30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).

Immunodetection and characterization of proteins implicated in renal sodium/phosphate cotransport

1994 ◽  
Vol 1190 (2) ◽  
pp. 289-296 ◽  
Author(s):  
Marie-Claude Delisle ◽  
Christian Boyer ◽  
Vincent Vachon ◽  
Sylvie Giroux ◽  
Richard Béliveau
Keyword(s):  
Sodium Phosphate ◽  
Phosphate Cotransport ◽  
Sodium Phosphate Cotransport

Sodium Phosphate Cotransport: Studies with Vesicles and LLC-PK1 Cells

Nephrology ◽  
1984 ◽  
pp. 57-69
Author(s):  
Heini Murer ◽  
Martin Amstutz ◽  
Jürg Biber ◽  
Piotr Gmaj ◽  
Kerstin Malmström
Keyword(s):  
Sodium Phosphate ◽  
Phosphate Cotransport ◽  
Sodium Phosphate Cotransport

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent α1-subunit of the Na+ pump

2006 ◽  
Vol 290 (4) ◽  
pp. R1142-R1150 ◽  
Author(s):  
Pedro Gomes ◽  
Patrício Soares-da-Silva
Keyword(s):  
Atpase Activity ◽  
Wild Type ◽  
Transfected Cells ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Functional Classes ◽  
A Cell ◽  
And Function ◽  
Α1 Subunit ◽  
Stimulation Of

Vectorial Na+ reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+ exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+ pump (Na+-K+-ATPase). We hypothesized that regulation of Na+ reabsorption should involve not only the activity of the basolateral Na+-K+-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na+-K+-ATPase, opossum kidney (OK) cells were transfected with the rodent Na+-K+-ATPase α1-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na+-K+-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na+-K+-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC50 values for ouabain: the first value was similar to the IC50 of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum α1-isozymes. It is shown that transfection of OK cells with Na+-K+-ATPase increased Na+-K+-ATPase and NHE3 activities. This was associated with overexpression of the Na+-K+-ATPase α1-subunit and NHE3 in transfected OK cells. The abundance of the Na+-K+-ATPase β1-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+ pump α1-subunit cDNA is expected to stimulate apical Na+ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.

Ontogeny of intestinal phosphate absorption in rabbits

1992 ◽  
Vol 262 (5) ◽  
pp. G847-G853 ◽  
Author(s):  
S. M. Borowitz ◽  
G. S. Granrud
Keyword(s):  
Brush Border ◽  
Sodium Phosphate ◽  
Brush Border Membrane ◽  
Phosphate Uptake ◽  
Membrane Vesicles ◽  
Phosphate Transport ◽  
Proximal Jejunum ◽  
Phosphate Cotransport ◽  
Distal Jejunum ◽  
Sodium Phosphate Cotransport

The ontogeny of intestinal phosphate transport was examined in brush-border membrane vesicles prepared from 2-wk, 4-wk, 6-wk, and 3-mo-old rabbits. At all four ages, vesicles prepared from the duodenum demonstrated sodium-phosphate cotransport in that uptake in the presence of a sodium gradient was significantly greater than uptake in the presence of a potassium gradient. While sodium-independent phosphate uptake was comparable at all ages, sodium-phosphate cotransport was greatest in the youngest animals and declined with increasing age. Peak phosphate uptake was 10-fold the equilibrium value at 2 wk and declined to 3-fold in adults. Maximal transport capacity of sodium-phosphate cotransport fell from 2,292 +/- 161 at 2 wk to 286 +/- 12 pmol.mg-1.10 s-1 at 3 mo, whereas the Michaelis constant did not change with age, varying between 0.032 and 0.054 mM. At all ages, uptake was half-maximally stimulated between 30 and 50 mM sodium, and Hill coefficients were between 1.5 and 2. Generation of inside negative diffusion potentials did not significantly enhance phosphate uptake at any age. These data suggest that throughout development, the transport of each phosphate molecule across the rabbit duodenal brush-border membrane is coupled to the transport of two sodium molecules. There were distinct developmental changes in the distribution of the sodium-phosphate cotransport along the length of intestine. In 2-wk old animals, sodium-phosphate cotransport was present in the duodenum, proximal and distal jejunum, and proximal ileum. In 4-wk-old animals, sodium-phosphate cotransport was present throughout the duodenum and proximal and distal jejunum, and in 6-wk and 12-wk-old animals, sodium-phosphate cotransport was observed only in the duodenum and proximal jejunum.

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