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J. Biol. Chem., Vol. 279, Issue 47, 49160-49171, November 19, 2004

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Partitioning of NaP_i Cotransporter in Cholesterol-, Sphingomyelin-, and Glycosphingolipid-enriched Membrane Domains Modulates NaP_i Protein Diffusion, Clustering, and Activity^*

Makoto Inoue, Michelle A. Digman¶, Melanie Cheng¶, Sophia Y. Breusegem||, Nabil Halaihel, Victor Sorribas**, William W. Mantulin¶, Enrico Gratton¶, Nicholas P. Barry||, and Moshe Levi||

From the Departments of Medicine, ^||Physiology, and Biophysics, Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado 80262, ^¶Laboratory for Fluorescence Dynamics, Department of Physics, University of Illinois, Urbana-Champaign, Illinois 61801, and the ^**Department of Toxicology, University of Zaragoza, E-50013 Zaragoza, Spain

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaP_i) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaP_i activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaP_i activity have not been determined. We determined if in potassium deficiency NaP_i is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaP_i in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaP_i partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaP_i was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaP_i protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaP_i protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaP_i protein is present in these microdomains, and that NaP_i lateral diffusion is slowed down and NaP_i aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/P_i cotransport activity in potassium deficiency.

Received for publication, August 4, 2004 , and in revised form, September 1, 2004.

^* This work was supported by a Veterans Affairs Merit Review, National Institutes of Health Grant 5R01 DK062209-02, Juvenile Diabetes Foundation Grant 1-2003-108 (to M. L.), the Cell Migration Consortium Grant PHSSUBUVGC 10641, National Institutes of Health Public Health Service Grant 5 P41-RRO3155, and by the University of Illinois, Urbana-Champaign (to E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

To whom correspondence should be addressed: University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80262. Tel.: 303-315-1541; Fax: 303-315-1929; E-mail: Moshe.Levi{at}UCHSC.edu.

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