title: Partitioning of NaPi cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched membrane domains modulates NaPi protein diffusion, clustering, and activity.
abstract: In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi 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 NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.
authors.affiliation: Department of Medicine, University of Colorado Health Sciences Center and Denver Veterans Affairs Medical Center, Denver, Colorado 80262, USA.
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.forename: Melanie
authors.initials: M
authors.lastname: Cheng
authors.forename: Sophia Y
authors.initials: SY
authors.lastname: Breusegem
authors.forename: Nabil
authors.initials: N
authors.lastname: Halaihel
authors.forename: Victor
authors.initials: V
authors.lastname: Sorribas
authors.forename: William W
authors.initials: WW
authors.lastname: Mantulin
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Barry
authors.forename: Moshe
authors.initials: M
authors.lastname: Levi
mesh_terms: D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004058:Diffusion; D006028:Glycosphingolipids / Q000737:chemistry*; D008055:Lipids / Q000737:chemistry; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism; D008853:Microscopy; D008871:Microvilli / Q000378:metabolism; D016011:Normal Distribution; D010710:Phosphates / Q000737:chemistry*; D017785:Photons; D011188:Potassium / Q000737:chemistry; D011191:Potassium Deficiency / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D013109:Sphingomyelins / Q000737:chemistry*; D027981:Symporters / Q000737:chemistry*; D014461:Ultracentrifugation
chemical_list: D003902:Detergents; D006028:Glycosphingolipids; D008055:Lipids; D010710:Phosphates; C493556:Slc34a1 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D013109:Sphingomyelins; D027981:Symporters; D002784:Cholesterol; D011188:Potassium; C018279:sodium phosphate