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Figure 1. . Voltage dependency of Pi activation in 100 mM Na+. (A) Pi activation characteristics for WT-expressing oocytes determined at six membrane potentials from â100 mV to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in ND100 + Pi and ND100 shown as mean ± SEM (n = 3). Currents were normalized to the magnitude of IPi at 1 mM Pi, V = â100 mV for each cell. Currents for these cells ranged from â76 to â88 nA at 1 mM, V = â100 mV. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 1. (B) Pi activation characteristics for G134C-expressing oocytes (n = 4) determined at six membrane potentials as in A, before (top) and after (bottom) incubation in 1 mM MTSEA for 3â5 min. Note the different ordinate scales for the âMTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS at 1 mM Pi, V = â100 mV. Currents for these cells ranged from â22 to â69 nA at 1 mM, V = â100 mV. (C) Pi activation characteristics for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3â5 min. Note the different ordinate scales for the âMTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C â MTS at 1 mM Pi, V = â100 mV. Currents for these cells ranged from â61 to â123 nA at 1 mM, V = â100 mV. (D) Voltage dependency of apparent Pi affinity (KmPi) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Pi activation data for the individual oocytes pooled in AâC. WT, filled circles; mutant â MTS, filled squares; mutant + MTS, empty squares. (E) Voltage dependency of predicted maximum Pi-dependent current (ImaxPi) for G134C (left) and M533C (right) with WT data superimposed. ImaxPi is shown as mean ± SEM predicted from fits to data for the individual oocytes pooled in AâC, with WT data superimposed. WT, filled circles; mutant â MTS, filled squares; mutant + MTS, empty squares. Maximum cotransport activity determined using the leak correction procedure (see text) is also shown. Mutant â MTS, filled triangles; mutant + MTS, empty triangles, with points joined by dashed lines.
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Figure 2. . Voltage dependency of Na+ activation in 1 mM Pi. (A) Na+ activation characteristics for WT-expressing oocytes determined at six membrane potentials from â100 to 0 mV as indicated by different symbols. Each data point is the difference between current recorded in NDX + Pi and NDX, shown as mean ± SEM (n = 4), where X is the respective Na+ concentration (in mM) and Pi = 1 mM. Currents were normalized to the magnitude of IPi at 1 mM Pi, V = â100 mV, and ND100. Arrow indicates direction of depolarization. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter. (B) Normalized Na+ activation data for G134C-expressing oocytes determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3â5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for G134C + MTS, and nH = 2 for G134C â MTS. Note the different ordinate scales for the âMTS and +MTS cases. Currents were normalized to the magnitude of IPi for G134C + MTS, ND100, V = â100 mV. (C) Normalized Na+ activation data for M533C-expressing oocytes (n = 4) determined at six membrane potentials as in A before (top) and after (bottom) incubation in 1 mM MTSEA for 3â5 min. Continuous lines are fits to the data points using Eq. 2 with nH as a free parameter for M533C â MTS, and nH = 2 for M533C + MTS. Note the different ordinate scales for the âMTS and +MTS cases. Currents were normalized to the magnitude of IPi for M533C â MTS, ND100, V = â100 mV. (D) Voltage dependency of apparent Na+ affinity (KmNa) for G134C (left) and M533C (right). Data points indicate mean ± SEM of KmPi reported from fits to Na+ activation data for the individual oocytes pooled in AâC. WT, filled circles; mutant â MTS, filled squares; mutant + MTS, empty squares.
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Figure 3. . Effect of MTS exposure on pH and voltage dependency. (A) Steady-state pHâV profile of WT showing IPi (1 mM total Pi) as a function of external pH and V. Currents were normalized to the magnitude of IPi at â100 mV, pH 7.4, and pooled (n = 4). Error bars omitted for clarity. Legend indicates color and corresponding IPi (norm). (B) Normalized, pooled (n = 5) IâV data for WT that compares IPi at pH 5.0 (open squares) with PFA-dependent current at pH 7.4 (filled triangles), normalized to the magnitude of IPi at â100 mV, pH 7.4 (filled squares). (C) Steady-state pHâV profiles of mutant G134C (left) and mutant M533C (right) showing IPi (1 mM total Pi) as a function of external pH and V before (âMTS) and after (+MTS) exposure to 1 mM MTSEA. Currents were normalized to the magnitude of IPi at â100 mV, pH 7.4, for G134C + MTS and M533C â MTS. Each data point is the mean of four cells from the same donor frog. Error bars omitted for clarity. The assay was repeated after exposure for 5 min to 1 mM MTSEA. Colored contour planes are as in A. (D) Voltage dependency of apparent inhibition constant for protons, KiH, (expressed in pH units) for G134C (left) and M533C (right) before (filled) and after (open) exposure to MTSEA. pKiH for WT is shown superimposed in both cases (filled circles). Data points were fitted with the modified Hill equation (Eq. 2) with nH as a free parameter and external proton concentration expressed logarithmically. Error bars indicate SEM of fit estimate for pKiH.
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Figure 4. . Effect of MTS exposure on presteady-state relaxations of mutants. (A) Representative presteady-state relaxations recorded from an oocyte expressing the WT NaPi-IIa protein superfused in 100 mM Na+ (ND100) and 0 mM Na+ (ND0) for the voltage step protocol shown in the inset. (B) Main component of presteady-state relaxation for a noninjected oocyte (NI) and the same WT-expressing cell in A, resolved by applying a two-exponential fitting procedure (see materials and methods). The ON (step from â60 mV to test potential) and OFF (step from test potential to â60 mV) relaxations are shown for superfusion in ND100 (top and middle traces) and ND0 (bottom traces). (C) Main ON and OFF relaxations for a representative G134C-expressing oocyte before (âMTS) and after (+MTS) exposure to 1 mM MTSEA (1 mM) for 3 min, superfused in ND100 and ND0. (D) Main ON and OFF relaxations for a representative M533C-expressing oocyte before (âMTS) and after (+MTS) exposure to 1 mM MTSEA (1 mM) for 3 min, superfused in ND100 and ND0.
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Figure 5. . Analysis of presteady-state relaxations; ON time constants (ÏON) for mutant G134C (A) and M533C (B). Voltage dependencies of time constants (ÏON) were obtained by fitting presteady-state relaxations in response to voltage steps from â60 mV to the indicated voltage with a double exponential (see materials and methods). Each point represents mean ± SEM of four cells. Fast ÏON (triangles), slow ÏON (squares); before MTSEA (filled symbols); after MTSEA (empty symbols) incubation (1 mM, 3 min). Continuous lines were obtained by fitting Eq. 4 to the data (Table I). Data for two superfusion conditions: ND100 (left) and ND0 (right). Data points at the holding potential (â60 mV) were determined from fits to the OFF relaxations.
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Figure 6. . Analysis of presteady-state relaxations: chargeâvoltage (QâV) data for a representative oocyte expressing G134C (A) and M533C (B). Each point is given by (QONâQOFF)/2, where QON and QOFF are the charges moved for the ON and OFF voltage steps from and to â60 mV, respectively. Errors smaller than symbol size are not shown. Left, superfusion in ND100; right, superfusion in ND0 for the same oocyte. Filled symbols, before MTSEA exposure; empty symbols, after MTSEA exposure. Dashed lines have been drawn to indicate the apparent equality of charge movement at hyperpolarizing potentials for ND100 and ND0 superfusion for G134C â MTS and M533C + MTS. Continuous lines were obtained by fitting Eq. 3 to the data. For G134C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.1/5.9 nC, Qhyp = â0.9/â3.4 nC; V0.5 = â7/â77 mV; z = 0.5/0.4; and in ND0 (±MTS), Qmax = 4.0/4.3 nC, Qhyp = â1.0/â2.1 nC; V0.5 = â5/â59 mV; z = 0.5/0.4. For M533C, the fit parameters were as follows: in ND100 (±MTS), Qmax = 5.8/3.7 nC, Qhyp = â2.0/â0.7 nC; V0.5 = â32/+11 mV; z = 0.6/0.6; and in ND0 (±MTS), Qmax = 4.8/2.9 nC, Qhyp = â2.1/â0.6 nC; V0.5 = â48/â5 mV; z = 0.5/0.5.
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Figure 7. . Modeling voltage-dependent presteady-state and steady-state kinetics for G134C â MTS and G134C + MTS. (A) Eight-state kinetic scheme for NaPi-IIa. Voltage-dependent transitions are shown bold. All other transitions are electroneutral. Critical transitions for simulating G134C â MTS (â) and G134C + MTS (+) are 1â8, 8â1, and 4â5. The corresponding voltage-independent rates used in the simulations are indicated. Dashed transition indicates a proposed leak pathway. (B) Simulations of presteady-state relaxations before (â) and after (+) MTS modification of Cys-134 using the model scheme of A for steps to potentials in the range â180 to +80 mV from Vh = â60 mV and two superfusion conditions. The vertical gray bars indicate the initial 2.5-ms interval after which presteady-state currents were resolved experimentally. Differential equations describing the transitions were solved for the state occupancies as a function of time. The presteady-state current per cotransporter molecule can be expressed as âe(αâ²(k12X1 â k21X2) â δ* (k18X1 â k81X8) + αâ²â²(k78X7 â k87X8)) (e.g., Parent et al., 1992), where Xn is the occupancy of state n and kij is the transition rate from state i to state j. The empty carrier (1â8) carries an equivalent charge of â1 that moves an equivalent electrical distance δ through the membrane. The Na+ binding transitions (1â2 and 8â7) also involve movement of +1 charge through equivalent electrical distances αⲠand αâ²â², respectively. To simplify the simulations, αâ²â² = 0.3, αⲠ= 0.3, and δ = 0.4 and the corresponding energy barriers were symmetrical. The rate constants for voltage-dependent transitions are given by k18 = k18o exp(δeV/2kT), k81 = k81o exp(âδeV/2kT), k12 = Na k12o exp(âαâ²eV/2kT), k21 = k21o exp(αâ²eV/2kT), where kijo is the rate constant for transition ij at V = 0, and Na is the concentration of Na+ (mM). Other fixed rate constants for the simulations were k12o = 2000 sâ1Mâ1, k21o = 600 sâ1, k23 = 7.5 à 106 sâ1Mâ1, k32 = 1000 sâ1, k34 = 106 sâ1Mâ2, k43 = 1000 sâ1, k54 = 25 sâ1, k78 = 100, k87 = 106 sâ1Mâ1, k67 = 1000 sâ1, k76 = 106 Mâ1sâ1, k65 = 105 sâ1Mâ2, k56 = 1000, k23= 0.005 sâ1. To satisfy microscopic reversibility, k32 and k76 were defined in terms of the other rate constants. (C) Predicted ÏâV data was obtained by applying a single exponential fit to the ON presteady-state currents for voltages in the range â180 to +60 mV, commencing 2.5 ms after the step onset. Filled symbols, âMTS; open symbols, +MTS; circles, ND100; squares, ND0. Model parameters are as given in A and B above for the âMTS and +MTS conditions with Pi = 0. (D) Predicted QâV data obtained by numerical integration of the presteady-state currents, commencing 2.5 ms after the step onset. Filled symbols, âMTS; open symbols, +MTS; circles, ND100; squares, ND0. Model parameters are as given in A and B above for the âMTS and +MTS conditions with Pi = 0. (E) Predicted steady-state IâV data for the âMTS (filled squares) and +MTS (open squares) conditions, using rate constants given in A and assuming Pi = 1 mM and external Na+ = 100 mM. The steady-state current is proportional to k14X1 â k41X4, i.e., the net flux for the only transition that involves transmembrane charge movement. Filled circles represent the simulated IâV data without changing k45 (see text). (F) Apparent substrate affinities for Pi activation (KmPi) (left) and Na+ activation (KmNa) (right) as a function of membrane potential. KmPi was obtained from a fit using the Michaelis-Menten equation (Eq. 1) to the steady-state activation data simulated with Pi in the range 0â3 mM and Na+ = 100 mM. KmNa was obtained from a fit using the modified Hill equation (Eq. 2) with Na+ in the range 0â125 mM and Pi = 1 mM. Filled symbols, âMTS; open symbols, +MTS. Error bars indicate ±SEM reported by fitting algorithm.
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Figure 8. . Simulations of occupancy of states 1, 2, 4, and 8 of the model scheme depicted in Fig. 7 A, as a function of membrane potential in zero external Na+ (ND0), 100 mM external Na+ (ND100), and 100 mM external Na+ with 1 mM Pi (ND100 + 1 mM Pi), before (â) and after (+) modification of Cys-134.
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