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Research findings indicate that urinary excretion of inorganic phosphate is regulated by the amount of the sodium/phosphate-cotransporter, NAPi-IIa, in the brush border membrane of proximal tubular cells. Data reveal that the amount of NaPi-IIa is dependent on the insertion rates of the newly synthesized cotransporters and by the endocytic rate. Furthermore, phosphate reabsorption is influenced by changes in the hormonally- and protein kinase regulated endocytic rate.

The transport function of the rat type IIa [Na.sup.+]/[P.sub.i] cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Set-460 lies in a functionally important region of the protein. To establish a 'structure-function' profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Set-460) showed a periodic accessibility pattern that would be expected for an [Alpha]-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated [Alpha]-helical structure. KEY WORDS: cysteine scanning * electrophysiology * mutagenesis * phosphate transport * secondary structure

I. INTRODUCTION: OVERALL MECHANISM Renal handling of [P.sub.i] determines its concentration in the extracellular space, the 'traffic' place between the two major body compartments: skeleton and intracellular space [37, 46, [...]

Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-[P.sub.i] cotransporter. Am J Physio! Renal Physiol 278: F792-F798, 2000.--Parathyroid hormone (PTH) inhibits proximal tubular reabsorption of [P.sub.i] by retrieval of type IIa Na-[P.sub.i] cotransporters (NaPi-IIa) from the brush-border membrane (BBM). We analyzed by immunohistochemistry whether PTH analogs, signaling through either protein kinase A (PKA) and C (PKC; 1-34 PTH) or only PKC (3-34 PTH), elicit in rat kidney in vivo or in the perfused murine proximal tubule in vitro a retrieval of NaPi-IIa and whether pharmacological agonists or inhibitors of these kinases are able to either mimic or interfere with these PTH effects. Treatment with either 1-34 or 3-34 PTH downregulated NaPi-IIa in rat kidney. In isolated murine proximal tubules 1-34 PTH was effective when added to either the apical or basolateral perfusate, whereas 3-34 PTH acted only via the luminal perfusate. These effects were mimicked by an activation of PKA with 8-bromoadenosine 3',5'-cyclic monophosphate or PKC with 1,2-dioctanoylglycerol. The luminal action of both PTH peptides was blocked by inhibition of the PKC pathway (calphostin C), whereas the basolateral effect of 1-34 PTH was completely abolished by inhibiting both pathways (H-89 and calphostin C). These results suggest that 1) NaPi-IIa can be internalized by cAMP-dependent and -independent signaling mechanisms; 2) functional PTH receptors are located in both membrane domains; and 3) apical PTH receptors may preferentially initiate the effect through a PKC-dependent mechanism. isolated proximal tubule; immunohistochemistry; parathyroid hormone receptor; type IIa sodium-phosphate transporter; protein kinase A; protein kinase C

Type II NaPi cotransporters mediate epithelial phosphate (Pi) reabsorption. In mammals the type IIb protein is expressed in the small intestinal apical membrane and other epithelia; it is not expressed in the renal proximal tubule where we find the type IIa isoform. To look for molecular determinant(s) involved in apical expression of type IIb cotransporters, we have made deletion mutations within the C-terminal tails of mouse IIb (mIIb) and human IIb (hIIb) transporter proteins. The constructs were fused to the enhanced green fluorescent protein and transiently transfected into intestinal CaCo2-cells. Both mIIb and hIIb were located exclusively in the apical membrane of the cells. For mIIb, the removal of a cysteine cluster or the last three amino acids (TVF) had no effect on the location of the protein. However, truncation at the level of the conserved L691/689 prevented the apical membrane expression of both mllb and hllb, respectively, and the mutated proteins were located in endosomal and lysosomal structures. A similar expression pattern of the mllb and hllb constructs was found in renal proximal tubular opossum kidney cells. Our data suggest that L691/689 is involved in mechanisms leading to an apical expression of type IIb NaPi cotransporters. epithelial polarity | enhanced green fluorescent protein

Traebert, Martin, Olaf Hattenhauer, Heini Murer, Brigitte Kaissling, and Jurg Biber. Expression of type II Na-[P.sub.i] cotransporter in alveolar type II cells. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L868-L873, 1999.--Type II Na-[P.sub.i] cotransporters (type IIa and type IIb) represent apically located Na-[P.sub.i] cotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-[P.sub.i] cotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-[P.sub.i] diet. After a chronic adaptation to a low-[P.sub.i] diet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-[P.sub.i] cotransporter in alveolar type II cells. sodium-inorganic phosphate cotransporter; immunohistochemistry

Hattenhauer, Olaf, Martin Traebert, Heini Murer, and Jurg Biber. Regulation of small intestinal Na-[P.sub.i] type IIb cotransporter by dietary phosphate intake. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G756-G762, 1999.--Dietary restriction of phosphate is a well-known stimulator (acting indirectly via vitamin [D.sub.3]) of small intestinal apical Na-[P.sub.i] cotransport. In the present study, we document by Western blots and immunohistochemistry that, in mice, a low-[P.sub.i] diet given for several days leads (in parallel to a stimulation of Na-[P.sub.i] cotransport) to an increase of the abundance of the type IIb Na-[P.sub.i] cotransporter in the brush-border membrane of mouse enterocytes. Similar results were also obtained by an injection of cholecalciferol. The abundance of the type IIb transcript was investigated by Northern blots. These results indicated that the amount of the type IIb transcript was not changed by either low-[P.sub.i] diet or cholecalciferol. It is concluded that stimulation of intestinal Na-[P.sub.i] cotransport by low-[P.sub.i] diet and vitamin D3 can be explained by an increased amount of type IIb Na-[P.sub.i] cotransporters in the brush-border membrane and that augmentation of type IIb Na-[P.sub.i] cotransporters is not related to an increased rate of transcription of the type IIb gene. Na-[P.sub.i] cotransport; intestine; adaptation; vitamin [D.sub.3]