HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kohl, B. Articles by Werner, A. Search for Related Content PubMed PubMed Citation Articles by Kohl, B. Articles by Werner, A.

The Na⁺-phosphate cotransport system (NaP_i-II) with a cleaved protein backbone: implications on function and membrane insertion

Beate Kohl *, Carsten A. Wagner ¹, Birgit Huelseweh *, Andreas E. Busch ¹ and Andreas Werner *

1. Renal handling of inorganic phosphate (P_i) involves a Na⁺-P_i cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaP_i-II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo.

2. The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N- (1-3 and 1-5) and C-terminal (4-8 and 6-8) complementary fragments of NaP_i-II from winter flounder. When the cognate fragments were used in combination (1-3 plus 4-8; 1-5 plus 6-8) they comprised the full complement of the putative transporter domains.

3. None of the four individual fragments or the 1-5 plus 6-8 combination when expressed in Xenopus oocytes increased P_i flux. Coexpression of fragments 1-3 plus 4-8 stimulated transport activity identical to that for expressed wild-type NaP_i-II with regard to pH dependency and K_m for Na⁺ and P_i binding; however, the maximal transport rate (v_max) was lower.

4. Immunohistochemistry on cryosections confined the functionally active 1-3 plus 4-8 combination to the oocyte membrane. This was not the case for the 1-5 plus 6-8 combination or any of the individual fragments, all of which failed to induce fluorescence.

5. A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N- nor C-terminal tags induced fluorescence.

This article has been cited by other articles:

				H. S. Tenenhouse and H. Murer Disorders of Renal Tubular Phosphate Transport J. Am. Soc. Nephrol., January 1, 2003; 14(1): 240 - 247. [Full Text] [PDF]

				N. A. WOLFF, B. GRUNWALD, B. FRIEDRICH, F. LANG, S. GODEHARDT, and G. BURCKHARDT Cationic Amino Acids Involved in Dicarboxylate Binding of the Flounder Renal Organic Anion Transporter J. Am. Soc. Nephrol., October 1, 2001; 12(10): 2012 - 2018. [Abstract] [Full Text] [PDF]

				A. Werner and R. K. H. Kinne Evolution of the Na-Pi cotransport systems Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2001; 280(2): R301 - R312. [Abstract] [Full Text] [PDF]

				H. Murer, N. Hernando, I. Forster, and J. Biber Proximal Tubular Phosphate Reabsorption: Molecular Mechanisms Physiol Rev, October 1, 2000; 80(4): 1373 - 1409. [Abstract] [Full Text] [PDF]

				H. Murer, I. Forster, N. Hernando, G. Lambert, M. Traebert, and J. Biber Posttranscriptional regulation of the proximal tubule NaPi-II transporter in response to PTH and dietary Pi Am J Physiol Renal Physiol, November 1, 1999; 277(5): F676 - F684. [Abstract] [Full Text] [PDF]

				S. Tatsumi, K.-i. Miyamoto, T. Kouda, K. Motonaga, K. Katai, I. Ohkido, K. Morita, H. Segawa, Y. Tani, H. Yamamoto, et al. Identification of Three Isoforms for the Na+-dependent Phosphate Cotransporter (NaPi-2) in Rat Kidney J. Biol. Chem., October 30, 1998; 273(44): 28568 - 28575. [Abstract] [Full Text] [PDF]

				K. Kohler, I. C. Forster, G. Stange, J. Biber, and H. Murer Identification of functionally important sites in the first intracellular loop of the NaPi-IIa cotransporter Am J Physiol Renal Physiol, April 1, 2002; 282(4): F687 - F696. [Abstract] [Full Text] [PDF]