The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2)

Abstract

The voltage dependence of the rat renal type II Na+/Pi cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (Pi) induced inward currents (Ip) in the presence of 96 mM Na+ over the potential range -140 </= V </= +40 mV. With Pi as the variable substrate, the apparent affinity constant (KmPi) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. KmPi increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum Ip (Ipmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, KmNa was weakly dependent on both Pi and voltage, the Hill coefficient was close to 3 and Ipmax was independent of Pi at -50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Na+ slippage. Voltage steps induced pre-steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V0.5) in the range -20 to -50 mV and an apparent valency of approximately 0.5 e-. V0.5 became more negative as Na+ was reduced. Pi suppressed relaxations in a dose-dependent manner, but had little effect on their voltage dependence. Reducing external pH shifted V0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step.

Figure 1

Voltage-dependent P_i-induced currents in an oocyte expressing the NaP_i-2 cotransporter. (A) Inward currents measured in response to the application of 1.0 mM P_i during the period indicated by the horizontal bar, at two holding potentials. Baseline current offsets have been suppressed to show P_i-induced current only. Arrow indicates time at which the staircase protocol (shown in B) is applied. Note that a similar fast change in current accompanies both application and washout of P_i. The ratio of initial fast component amplitude to the steady state current was the same for both potentials and depended on the superfusion conditions. The slower component possibly reflects the rate of P_i diffusion through the unstirred layer of oocyte that is not directly exposed to the superfusate. The cell was bathed in standard ND96 solution (see materials and methods). (B) Response of another oocyte expressing NaP_i-2 to voltage staircase protocol applied ∼17 s after application of P_i (A, arrow). Each step corresponds to a −20-mV membrane potential change. Same superfusion conditions as in A. (top traces) Response to voltage staircase, from −140 to +40 mV, superimposed on −50-mV holding potential (V_h) in the absence (−P_i) and presence (+P_i) of 1 mM P_i. Note that the steady state current immediately before and after the staircase is the same in each case. The current spike at each voltage transition results from incomplete cancellation of the endogenous capacitive transient and pre–steady state relaxations using the capacitive transient simulator (see materials and methods). (bottom) P_i-induced current obtained by subtraction of the two traces. Dashed line indicates zero holding current level.

Figure 2

Steady state voltage dependency of Na⁺/P_i transport as a function of P_i. (A) Typical set of I-V curves obtained using staircase protocol (Fig. 1 B) applied to one cell with 96 mM Na⁺ and P_i in the range 0.006–1.0 mM as indicated. Data points are joined for visualization only. Note the absence of zero crossing for V > 0. (B) Representative dose-response curves at V_h = −50 mV for two concentrations of Na⁺: 96 (▪), 50 (□) mM and the same cell with P_i as the variable substrate. Eq. 1 was fit to the raw data with the following parameters: (96 mM Na⁺) K _m ^Pi = 0.054 mM, n = 0.8, I_pmax = 74 nA; and (50 mM Na⁺) K _m ^Pi = 0.27 mM, n = 0.9, I_pmax = 50 nA. Inset shows the same data normalized to maximum predicted response and plotted semi-logarithmically to indicate clearly the shift in K _m ^Pi. (C) Summary of voltage dependency of fitted parameters comparing data for two concentrations of Na⁺: 96 (▪) and 50 (□) mM pooled from different cells. Data are shown as mean ± SEM (N = 4). Only SEMs exceeding symbol size are shown. Data points are joined for visualization only. (C, 1) n = Hill coefficient, (dashed line) n = 1; (C, 2) K _m ^Pi = apparent affinity constant; (C, 3) I_pmax/I_pmax(−100) = maximum induced current normalized to the value at −100 mV.

Figure 3

Steady state voltage dependency of Na⁺/P_i transport as a function of Na⁺ concentration. (A) Typical family of I-V curves obtained from one cell with 1 mM P_i and six Na⁺ concentrations as indicated (millimolar). Data points are joined for visualization only. (B) Typical dose-response data for the same cell at two P_i concentrations: 1.0 (▪) and 0.1 (□) mM with Na⁺ as the variable substrate and V_h = −50 mV. Eq. 1 was fit to the data points. For this cell, the fit parameters were: (1 mM P_i) K _m ^Na = 50.1 mM, n = 2.6, I_pmax = 109 mM; and (0.1 mM P_i) K _m ^Na = 89 mM, n = 2.6, I_pmax = 114 mM. (C) Summary of voltage dependence of fitted parameters comparing data for two concentrations of P_i: 1 (▪) and 0.1 (□) mM. Data are shown as mean ± SEM. Only SEMs exceeding symbol size are shown. Data points are joined for visualization only. (C, 1) n = Hill coefficient, (dashed line) n = 3; (C, 2) K _m ^Na = apparent affinity constant; (C, 3) I_pmax/I_pmax(−100) = maximum induced current normalized to the value at −100 mV.

Figure 4

Characterization of P_i-independent current component using phosphonoformic acid. (A) Oocyte holding current (I_h) at V_h = −50 mV for continuous superfusion with the indicated solutions for a NaP_i-2-expressing oocyte (top) and noninjected oocyte (bottom) from the same batch of oocytes and recorded during the same experimental session. I_h was sampled at 5-s intervals and the sample points have been joined by straight lines for clarity. Each superfusate combination was applied for 1 min to allow a stable baseline to be reached. Top and bottom dashed lines, superimposed on NaP_i-2 data, indicate I_h at 9 and 105 mM Na⁺, respectively. (B) Typical I-V relations for the PFA-sensitive component at two concentrations of Na⁺: 109 (▪) and 59 (▴) mM. A staircase voltage protocol with 5-mV, 100-ms-long steps was applied to the oocyte. Points represent the difference between the steady state current at the end of each step under control conditions and the response in the presence of 3 mM PFA. As PFA is a trisodium salt, the control solution Na⁺ concentration in each case was adjusted to ensure that the Na⁺ gradient remained the same. Continuous lines are polynomial fits to the data, used to determine the reversal potential (E_r). Inset shows E_r plotted as a function of Na⁺ concentration. Note that Na⁺ concentration is plotted on a log₁₀ scale. Number of cells is indicated for each Na⁺ tested. Straight line is a linear regression giving a slope 64.4 ± 1.7 mV. (C) Dose dependency with respect to Na⁺ for I_h in the absence of P_i at V_h = −50 mV. Data shown for a typical cell expressing NaP_i-2. Each point represents the induced change in steady state current, relative to 0 mM Na⁺. Continuous curve is the fit using Eq. 1, giving K _m = 128 mM, I_hmax = 74 nA, and n = 0.92. (D) The PFA-sensitive current correlates with the P_i-induced current. Data shown for 22 cells from several donor frogs displaying different levels of expression of NaP_i-2. For each cell, the P_i-induced current (I_p) at 1 mM P_i was determined together with the PFA-sensitive component (I_PFA) for 3 mM PFA at V_h = −50 mV. The straight line is a linear regression line forced through the origin with a slope: 0.126 ± 0.004.

Figure 5

Pre–steady state relaxations induced by voltage steps applied to oocytes expressing NaP_i-2. (A) Family of current records in response to voltage steps from V_h = −100 to −140, −120, −80, −40, 0, +40, and +80 mV, and returning to V_h after 40 ms. (top) Superfusion with 96 mM Na⁺, 0 mM P_i. The endogenous capacitive charging transients of the oocyte have been clipped due to the high voltage clamp gain used. (middle) Superfusion with 96 mM Na⁺, 3 mM P_i. Dashed line, corresponding to the holding current level at −100 mV in the absence of P_i, is superimposed to indicate the change in steady state current induced by P_i (at −100 mV, I_p ≈ 250 nA). (bottom) Superfusion with 96 mM Na⁺, 0 mM P_i for a noninjected oocyte from the same batch. Each record is the average of eight successive raw sweeps. Filtering at 3 kHz, sampling 50 μs/point. (B) Effect of PFA pre–steady state currents. Records in response to a voltage step from −100 to 0 mV for superfusion in the presence of 0.3 mM P_i or 3 mM PFA compared with the response for 96 mM Na⁺ alone, as indicated. Dashed line indicates baseline for control condition. Each record is the average of eight successive raw sweeps and has been blanked for 2 ms during the capacitive charging transient. Filtering at 3 kHz, sampling 50 μs/point. (C) Voltage dependency of the main time constant (τ) obtained from biexponential fit to records such as in A for holding potentials (V_h) −100, −40, and 0 mV for the same cell. Filled symbols are mean ± SEM of the ON transition τs at the three V_h. The straight lines represent the mean of the OFF relaxation τs over the whole voltage range: (dotted line) V_h = −100 mV, (dashed line) V_h = 0 mV. The mean OFF τs are: 7.5 ± 0.06 ms (V_h = −100 mV); 7.6 ± 0.05 ms (V_h = −40 mV), and 7.13 ± 0.14 ms (V_h = 0 mV). (D) Voltage dependency of charge movement (Q) associated with NaP_i-2-related component at different V_h for the same cell as in B. (▪) ON, (□) OFF. Continuous lines are fits of the Boltzmann function (Eq. 2) to the data. See Table I for fit parameters. (E) Correlation between estimated charge available for translocation at −100 mV (Q₍₋₁₀₀₎) and P_i-induced current at −100 mV (I_p(−100)) with 96 mM Na⁺ and 1 mM P_i. Data points are from 11 cells from different oocyte batches. Q₍₋₁₀₀₎ was obtained by fitting the Boltzmann function (Eq. 2) to the Q-V data and I_p(−100) was obtained from steady state response of same cell under same recording conditions. Straight line is a linear regression line with slope 46 s⁻¹ and forced to intercept the origin.

Figure 7

The effect of changing external P_i on pre–steady state relaxations. (A) Pre–steady state relaxations recorded from a cell expressing NaP_i-2 for a voltage jump from −100 to 0 mV for 0, 0.01, 0.03, 0.06, 0.1, 0.3, 1, and 3.0 mM P_i. The initial baseline shift reflects the steady state holding current induced by each P_i. P_i was applied in increasing concentration. Between successive applications, the cell was allowed to recover in 0 mM P_i until the initial steady state holding current at −50 mV was reestablished. (B) The P_i-suppressed current obtained by subtracting the record at 3 mM P_i from the test record under the same voltage step conditions as A and same cell. Each trace has been baseline-adjusted to the steady state value. The first millisecond of each difference record during the voltage transition was blanked. This corresponds to the time to complete most of the oocyte capacitive charging as indicated in the inset for a voltage jump from −50 to −40 mV, plotted on the same time scale (same cell). (C) P_i dose response for the same cell showing amount of apparent charge suppressed (Q_{suppr .}) at −100 mV as a function of P_i for five target potentials as indicated. Charge was estimated by numerical integration of the records as in B, with the steady state holding current suppressed. A small error is expected in Q_suppr. due to starting the integration at 1 ms. Continuous lines show fits with Eq. 1 for n = −1. (D) Voltage dependence of apparent K _d for suppression of pre–steady state charge found from fitting Eq. 1 to the data, for the same cell as in C.

Figure 6

The effect of changing external Na⁺ on pre–steady state relaxations. (A) Typical response to a voltage step from −100 to 0 mV for a cell expressing NaP_i-2 (I_p = −120 nA at −50 mV, 3 mM P_i). The cell was superfused with 96, 50, 25, and 0 mM Na⁺ and sufficient time was allowed between changing superfusate to establish a steady holding current before making pre–steady state recordings. The pre–steady state response to superfusion with 96 mM Na⁺ and 3 mM P_i is also shown. For superfusion with 0 mM Na⁺ and 3 mM P_i, the response was the same as for 0 mM Na⁺ alone (data not shown). All traces were blanked for the first 2 ms after the step and adjusted to give the same steady state baseline. (B) Steady state charge distribution under varying Na⁺. The Q-V data (ON transition) for a typical cell with superfusion with 96 (▪), 50 (▴), 25 (•), and 10 (♦) mM Na⁺. Continuous lines were obtained by fitting the Boltzmann function (Eq. 2) to the data. Inset shows the same data normalized to the predicted maximum charge and offset to superimpose at the depolarizing limit to indicate the shift in V_0.5. (C) τ-V data pooled (n = 4) showing main ON relaxation time constant determined for superfusion with 96 (□), 50 (▵), and 25 (○) mM Na⁺.

Figure 8

The effect of external pH on pre–steady state relaxations. (A) Superimposed pre–steady state relaxations for ON and OFF voltage steps to the test potentials indicated from a holding potential of −100 mV for the same cell with 96 mM Na⁺ in external medium. Thick traces, pH 7.4; thin traces, pH 6.2. To suppress the endogenous capacitive transient, the records were blanked for the first 3 ms after the transition. (B) Apparent charge movement for three pHs indicated in the presence of 96 mM Na⁺. Continuous lines are fits using the Boltzmann function (Eq. 2). The fit parameters were: (pH 7.4) z = 0.38, V_0.5 = −40 mV, Q_max = 7.4 nC; (pH 6.8) z = 0.37, V_0.5 = −23 mV, Q_max = 7.5 nC; (pH 6.2) z = 0.46, V_0.5 = −2 mV, Q_max = 5.3 nC. (C) Voltage dependence of main ON relaxation τ for three pHs indicated and the same cell as in B. (D) Pre–steady state relaxations recorded from another cell for a step from −100 to 0 mV (ON) and returning to −100 mV (OFF) for three superfusion conditions: 96 mM Na⁺, pH 7.4 (thin trace); 0 mM Na⁺, pH 7.4, and 0 mM Na⁺, pH 6.2 (arrows). The graphical superposition of the traces reveals that after removal of Na⁺ from the external medium, a reduction in pH leads to a further suppression of the relaxations. To suppress the endogenous capacitive transient, the records are blanked for the first 3 ms after the transition.

Scheme I

Figure 9

A state diagram representation of an ordered binding model for NaP_i-2. Voltage-dependent partial reactions identified by pre–steady state experiments (translocation of the unloaded carrier, 6 ⇔ 1; binding of the first Na⁺, 1 ⇔ 2) are shown shaded. The empty cotransporter is assumed to have a valency of −1, but this charge translocates through a fraction of the transmembrane field to account for an apparent valency <1 derived from fits using Eq. 2. Both the slippage (2 ⇔ 5*) pathway and translocation of the fully loaded carrier (4 ⇔ 5) are assumed to be electroneutral. Depending on the availability of substrate and membrane potential, accessibility to the substrate binding sites favors either the cis (states 1, 2, 3, 4) or trans (states 5, 5*, 6) side of the membrane. Under normal physiological conditions with V < 0, the apparent transport cycle proceeds clockwise around the loop. For V < 0, and low internal Na⁺, external Na⁺ binding is facilitated on the cis side due to the outward translocation of −ve charge associated with transition 6 ⇔ 1. This allows one Na⁺ ion to move to its binding site within the transmembrane field, leading to an increasing of the affinity of the transporter for P_i (assumed to be predominantly divalent at neutral pH), which then binds (2 ⇔ 3) in a voltage-independent manner. Then follows a further voltage-independent step involving the binding of two Na⁺ ions (3 ⇔ 4) to give electroneutrality. The fully loaded carrier is now able to translocate (4 ⇔ 5), release substrates on the trans side, and return to state 6. A net inward charge movement of +1 electronic units occurs per cycle, which is manifested as I_p. Except for slippage, the order of binding/release of substrates on the cytosolic side cannot be determined using the intact oocyte preparation and, therefore, the cytosolic release of cotransported P_i, together with two Na⁺ ions, is lumped as one reaction (5 ⇔ 5*).

Figure 10

Simulations predict voltage-dependent behavior of pre–steady state kinetics in the absence of external P_i. Values were assigned to the parameters associated with voltage-dependent partial reactions (shown shaded in Fig. 9: unloaded carrier, 6 ⇔ 1; first Na⁺ binding/debinding, 1 ⇔ 2) to give a reasonable match to the measured τ-V and Q-V data under varying external Na⁺. (A) Simulations of pre–steady state current showing the ON and OFF relaxations for a step from −100 to 0 mV for two external Na⁺ concentrations (continuous lines, 100 mM; broken lines, 50 mM). The voltage step is assumed to occur instantaneously so that no account is taken of the speed of oocyte membrane charging. Note that because of the voltage dependence of k₂₁, the fast component in the relaxation appears more prominent in the ON than OFF traces for both Na⁺, and this can lead to significant errors in estimating Q_ON and Q_OFF (see discussion). Rate constants for voltage dependent steps are (s⁻¹): k₆₁ = 60 exp(−0.16 eV/kT), k₁₆ = 120 exp(0.24 eV/kT), k₁₂ = 8,000 [Na] exp(−0.15 eV/ kT), k₂₁ = 2,000 exp(0.15 eV/kT), where [Na] = Na⁺ concentration (Molar). The corresponding valences and asymmetry factors are: z₆₁ = 0.4, z₁₂ = 0.5, δ₆₁ = 0.4, δ₁₂ = 0.5. Ordinate scale is in electronic units (eu) s⁻¹, where 1 eu = 1.602 × 10⁻¹⁹ C. (B) Simulated τ-V relations for two external Na⁺ concentrations as in A. Bold curves represent the two nonzero time constants predicted from the eigenvalue solutions of the three-state model involving transitions 6 ⇔ 1 and 1 ⇔ 2 (continuous curves, 100 mM; broken curves, 50 mM). The faster component with τ < 1 ms would not be detected easily by curve fitting due to the oocyte charging transient. Light curves represent the voltage dependence of the reciprocal rate constants for the transitions indicated by the respective subscripts. Note that only k₁₂ is dependent on Na⁺. (C) Simulated steady state Q-V relation for the same two Na⁺ concentrations. The amount of charge in electronic units contributed by the two transitions is shown (Q₆₁, Q₁₂), together with the total charge (Q_t). For the simulation, the holding potential was set at −1,000 mV to obtain normalized relations. Fitting Eq. 2 to the Q_t-V data predicted z = 0.5 and a shift of −16 mV for a change in Na⁺ from 100 to 50 mM. (D) Simulation of the effect of pH on pre–steady state kinetics. The unloaded carrier backward rate constant (K₁₆) is assumed to be decreased from 120 to 60 s⁻¹, resulting from an increase in external H⁺ as indicated by measurements. The τ-V curves predict an increase of the slower relaxation for V > 0. Continuous lines represent τs (bold) and inverse rate constants (light) under normal (pH 7.4) conditions. Broken lines are for reduced pH conditions. (E) The corresponding steady state Q-V relations for the same change in rate constant K₁₆ showing the shift in the Q-V distribution towards depolarizing potentials (broken lines) resulting primarily from a shift in the steady state charge distribution of the unloaded carrier.

Figure 11

Simulations of P_i-induced currents in the steady state, assuming zero trans conditions. Since the transition 6 ⇔ 1 is the only translocation step involving net charge movement, the steady state current is proportional to X₁k₁₆ − X₆k₆₁, where X₁ and X₆ are the occupancy of states 1 and 6, respectively. Voltage-dependent rate constants and parameters are given in Fig. 10. Additional rate constants for the kinetic scheme of Fig. 9 are (s⁻¹): k₂₃ = 1,000 [P_i], k₃₂ = 100, k₃₄ = 500 [Na]², k₄₃ = 50, k₄₅ = k₅₄ = 25, k_55* = 10,000, k_5*5 = 0, k_5*6 = 10,000, k_65* = 0, k_25* = 2.5, k_5*2 = 2.5, where [P_i] and [Na] are the concentrations of P_i and Na⁺, respectively (Molar). These were chosen to give reasonable predictions of the experimentally observed I-V relations. (A) P_i dose response. Inset shows a set of I-V curves for nominal P_i values indicated, normalized to I_p at −100 mV. Dashed curve is the slippage component (simulated with P_i = 0 mM), light curves represent the total simulated P_i-induced steady state current and bold curves represent the steady state response with slippage component subtracted (equivalent to the P_i-induced response measured). Eq. 1 was fit to the data to obtain the apparent K _m ^Pi as a function of V for nominal 100 (▪) and 50 (□) mM Na⁺. The continuous lines are for visualization only. (B) Na⁺ dose response. (inset) Set of I-V curves for nominal Na⁺ values indicated, and 1 mM nominal P_i, normalized to I_p at −100 mV. Light curves represent the simulated total steady state current, bold curves have slippage component subtracted (equivalent to the P_i-induced response measured). Eq. 1 was fit to the data to derive the apparent K _m ^Na as a function of V for nominal 1 (▪) and 0.1 (□) mM P_i. The continuous lines are for visualization only.