title: Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step.
abstract: The SLC34 family of Na(+)-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na(+)/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na(+) ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na(+) ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na(+) ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na(+) to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate- and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetics.
authors.affiliation: Institute of Physiology and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Monica
authors.initials: M
authors.lastname: Patti
authors.forename: Chiara
authors.initials: C
authors.lastname: Ghezzi
authors.forename: Ian C
authors.initials: IC
authors.lastname: Forster
mesh_terms: D000200:Action Potentials; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D014981:Xenopus
chemical_list: D010710:Phosphates; C493563:Slc34a3 protein, mouse; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc; D012964:Sodium