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Identification of the First Sodium Binding Site of the Phosphate Cotransporter NaPi-IIa (SLC34A1)

Lookup NU author(s): Dr Andreas Werner


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Transporters of the SLC34 family (NaPi-IIa,b,c) catalyze uptake of inorganic phosphate (P-i) in renal and intestinal epithelia. The transport cycle requires three Na+ ions and one divalent P-i to bind before a conformational change enables translocation, intracellular release of the substrates, and reorientation of the empty carrier. The electrogenic interaction of the first Na+ ion with NaPi-IIa/b at a postulated Na1 site is accompanied by charge displacement, and Na1 occupancy subsequently facilitates binding of a second Na+ ion at Na2. The voltage dependence of cotransport and presteady-state charge displacements (in the absence of a complete transport cycle) are directly related to the molecular architecture of the Na1 site. The fact that Li+ ions substitute for Na+ at Na1, but not at the other sites (Na2 and Na3), provides an additional tool for investigating Na1 site-specific events. We recently proposed a three-dimensional model of human SLC34a1 (NaPi-IIa) including the binding sites Na2, Na3, and P-i based on the crystal structure of the dicarboxylate transporter VcINDY. Here, we propose nine residues in transmembrane helices (TM2, TM3, and TM5) that potentially contribute to Na1. To verify their roles experimentally, we made single alanine substitutions in the human NaPi-IIa isoform and investigated the kinetic properties of the mutants by voltage clamp and P-32 uptake. Substitutions at five positions in TM2 and one in TM5 resulted in relatively small changes in the substrate apparent affinities, yet at several of these positions, we observed significant hyperpolarizing shifts in the voltage dependence. Importantly, the ability of Li+ ions to substitute for Na+ ions was increased compared with the wild-type. Based on these findings, we adjusted the regions containing Na1 and Na3, resulting in a refined NaPi-IIa model in which five positions (T200, Q206, D209, N227, and S447) contribute directly to cation coordination at Na1.

Publication metadata

Author(s): Fenollar-Ferrer C, Forster IC, Patti M, Knoepfel T, Werner A, Forrest LR

Publication type: Article

Publication status: Published

Journal: Biophysical Journal

Year: 2015

Volume: 108

Issue: 10

Pages: 2465-2480

Print publication date: 19/05/2015

Online publication date: 19/05/2015

Acceptance date: 17/03/2015

ISSN (print): 0006-3495

ISSN (electronic): 1542-0086

Publisher: Cell Press


DOI: 10.1016/j.bpj.2015.03.054


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Funder referenceFunder name
NIH, National Institute of Neurological Disorders and Stroke
Swiss National Science Foundation
Hartmann Muller Foundation
AW SA10/0210Dunhill Medical Trust