Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

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.