PMC

Current-voltage (I-V) data for the WT and functional mutants with substitutions at sites in TM2. Each panel shows the normalized P_i-dependent current (I_Pi) under four superfusion conditions: 100Na + 1 mM P_i (black squares); 100Na + 0.1 mM P_i (gray squares), 50Na + 1 mM P_i (blue circles), and 50Na50Li + 1 mM P_i (red circles). Data were pooled from more than three batches of oocytes and each point represents the mean ± SE of n > 10 oocytes. Data for each oocyte were normalized to the control response to 1 mM P_i in 100Na at −100 mV. Data points are joined for graphical clarity.

Steady-state properties of TM2 mutants. (A) Normalized I-V data for WT and mutants. Each data set is the I_Pi for 1 mM P_i in 100 Na normalized to the response at −100 mV. (Left) WT and mutants involving substitution of polar residues. (Right) WT and mutants involving substitution of acidic or basic residues. Data points have been joined for graphical clarity. (B) The effect of cation replacement at −100 mV is depicted as the ratio of I_Pi with 1 mM P_i superfusion of either 50Na + 1 mM P_i (blue bars) or 50Na50Li + 1 mM P_i to the control response in 100 Na⁺ 1 P_i (red bars). Each bar shows the mean ratio ± SE for n > 5 oocytes expressing the indicated construct.

Refined structural model of human NaPi-IIa bound to three Na⁺ ions and one P_i anion. The Na⁺ ions are shown as purple spheres and the P_i is shown as orange and red spheres. Residues comprising RU1 and RU2 are colored in green and pink, respectively. (A) Close-up of the Na3-binding site, viewed from the extracellular side of the membrane into the binding site. The Cα atoms of several residues predicted to form the Na3 site or to coordinate P_i are highlighted (spheres). (B) Predicted Na1-binding site. The Cα atoms of the residues whose side chain and/or backbone atoms are predicted to form the Na1 site are shown as green spheres. The Cα atoms belonging to residues R210 and D224 are highlighted as light blue spheres. (C) Overview of the human NaPi-IIa model represented as cartoon helices, viewed from along the plane of the membrane.

Candidate Na1-binding-site residues in a published structural model of human NaPi-IIa based on VcINDY. The model included two Na⁺ ions (purple spheres) and one P_i anion (yellow and red sticks), and was generated using the alignment described previously (). The protein backbone is represented as cartoon helices with RU1 colored green and RU2 in pink, and is viewed from along the plane of the membrane from the face furthermost from the dimer interface in the template. TM2b and TM5a are colored blue and cyan, respectively. Candidate Na1-binding-site residues are represented as spheres at their Cα positions. Residues whose mutation had an effect on transport activity, apparent affinities for substrate or ions, or steady-state currents are colored red or pink. The residue whose mutation did not alter any of these parameters (T211) is colored blue. Residues D224 and N227 are colored gray, and their side chain and Cα atoms are represented as sticks and spheres, respectively.

Quantification of midpoint voltage (V_0.5) for WT and mutants. (A) Comparison of V_0.5 for superfusion in 100Na (solid bars) and 0Na (open bars) for each construct. Each bar represents the mean ± SE for n ≥ 4 cells from at least two donor animals. (B) Dependence of V_0.5 on external [Na⁺] for mutants of two neighboring charged sites (D209 and R210) in TM2. Data points are joined by lines. (C) The same data as in (B) plotted on a semilog scale. The continuous lines are best fits to the data points predicted by a four-state model that accounts for the empty carrier and sequential Na⁺ binding partial reactions (see Discussion). The dashed line represents the theoretical limiting slope of 116 mV/decade expected at high [Na⁺].

Presteady-state relaxations. (A) Representative examples of presteady-state relaxations evoked by voltage steps in the range of −180 mV to +80 mV from a −60 mV holding potential for a typical noninjected (NI) oocyte, the WT, and mutants D209E and T454A. Upper data traces correspond to superfusion in 0 Na solution, and lower traces correspond to superfusion in 100Na solution. For each construct, Q-V data are shown for the two superfusates. Gray dashed lines help to visualize the charge distribution about the holding potential. Continuous lines are fits to a single Boltzmann function (Eq. 1). The parameters for the fits for the individual cells shown are summarized in . (B) Ratio of the estimated total charge predicted from the Boltzmann equation fit to data obtained by superfusion with 0Na to that obtained by superfusion with 100Na (Q_max⁰/Q_max¹⁰⁰) for each construct. Data are shown as mean ± SE for n > 5 cells. Black, WT; gray, mutants in TM2; white, mutants in TM5.

Kinetic scheme for the electrogenic NaPi-IIa/b isoforms. Schematics represent the conformational states identified by previous functional analyses to show the occupancy of the proposed substrate-binding sites. The physiologically relevant cotransport cycle (clockwise, starting from state 0) involves a voltage-dependent reorientation of the unloaded carrier (state 0) to state 1 (assuming a hyperpolarized membrane potential), and movement of a single Na⁺ ion to the proposed Na1 site (state 2), followed by the cooperative interaction of a second Na⁺ ion before divalent P_i and a final Na⁺ ion, resulting in occupancy of the Na2-P_i-Na3 sites (state 4). A major translocation event then occurs (transition 4–5) and substrates can be released to the cytosol (transition 5–6). The last Na⁺ unbinding event (transition 6–0) effectively frees the empty carrier intrinsic charge to allow reorientation of the empty carrier to state 1 under the influence of a hyperpolarizing TM potential, in readiness for the next cycle. For the electroneutral NaPi-IIc, the empty carrier reorientation is voltage independent and no detectable charge displacement is associated with any of the partial reactions.