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Figure 1. Topology model and basic characterization of the S448C mutant. (A) Topology model predicting eight transmembrane segment and two reentrant loops, which dip into the membrane. N and C termini are intracellular. The site of the Ser-448-Cys mutation is indicated. (B) Voltage dependency of apparent affinity constant for Pi interaction (\documentclass[10pt]{article}
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\begin{equation*}K_{{\mathrm{m}}}^{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document}), determined at 100 mM Na+ for WT and S448C. Each data point is the mean ± SEM of the \documentclass[10pt]{article}
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\begin{equation*}K_{{\mathrm{m}}}^{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document} estimated from n = 4 oocytes. (C) Voltage dependency of the apparent affinity constant for Na+ interaction (KmNa), determined at 1 mM Pi for WT and S448C. Each data point is mean ± SEM of the KmNa estimated from n = 3 oocytes. (D) Original current traces obtained from an oocyte expressing S448C in response to voltage jumps between −120 and +60 mV from a holding potential of −60 mV in ND100 solution (left) or ND100 + 1 mM Pi (right). Upper traces were acquired before, and lower traces after the oocyte was exposed to MTS-TMR for 5 min. (E) Current–voltage relationship of S448C obtained by subtracting recordings similar to those shown in D before and after labeling with tetramethylrhodamine-methanethiosulfonate (MTS-TMR). Data points were normalized to \documentclass[10pt]{article}
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\begin{equation*}I_{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document} at −100 mV (n = 4).
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Figure 2. Time course of modification of Cys-448 by MTS reagents. (A) Original voltage (top) and current (bottom) trace acquired from an oocyte expressing S448C and repeatedly exposed to 25 μM MTSEA for successive 1-min intervals (white bars). During MTSES exposure, the membrane potential was held at −90 mV. After washout of MTSES, the membrane potential was changed to −50 mV and 1 mM Pi was applied (gray bars) to record the Pi-induced current. (B–D) \documentclass[10pt]{article}
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\begin{equation*}I_{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document}, as a function of cumulative exposure time in MTSES (B), MTSEA (C), and MTSEA in the absence of Na+ (ND0 superfusate) (D). Abscissa is the cumulative exposure time (t) at the indicated incubation holding potential. In each panel, \documentclass[10pt]{article}
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\begin{equation*}I_{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document} (at Vh = −50 mV) after each MTS exposure was normalized to \documentclass[10pt]{article}
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\begin{equation*}I_{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document} at t = 0 and fitted with Eq. 3 (solid line) to obtain the effective second-order rate constant (see Table I).
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Figure 3. Steady-state fluorescence measurements. (A) Original trace showing the fluorescence signal F and the holding current I recorded on the Minidigi 1A from an experiment in which the Na+ concentration was varied. The S448C-expressing and MTS-TMR–labeled oocyte was initially clamped at −60 mV and superfused with ND100 solution. After we equilibrated the oocyte with a new test solution, the shutter was opened periodically for seven successive 230-ms pulses followed by a 2-s closing time. To control for fluorescence rundown, each test solution was bracketed by ND100 solution. For better visualization of the rundown, the ND100 data is connected by a dotted line (drawn by eye). During the shutter opening, the membrane potential was stepped from −60 to −120 mV. These step changes in membrane potential are the cause of the capacitive current spikes visible in the current recording. Note the change in holding current as the Na+ concentration is changed. When the shutter is closed, F is outside the measurable range. (B) ΔF/F plotted as a function of Na+ for a representative oocyte, after correction for fluorescence rundown. Continuous lines are fits to data using the Hill equation. (C) ΔF/F plotted as a function of Li+ for a representative oocyte using a similar protocol as in A but with variable Li+ instead of Na+. Data were fitted with the Hill equation with H constrained to 1. (D) ΔF/F plotted as a function of Pi with Na+ constant at 100 mM. During the shutter opening time, the membrane potential was stepped from −60 to 0 mV. Data for individual oocytes were fit with the Hill equation (Eq. 1) with H constrained to 1, normalized to ΔFmax at 0 mV, pooled, and refit with Eq. 1 (n = 5). See text for all fit parameters.
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Figure 4. Cation dependency of the voltage-dependent fluorescence in oocytes expressing S448C. (A) Original fluorescence trace recorded in ND100 (top), ND0 (middle), and LD100 (bottom) solutions from an oocyte labeled with MTS-TMR. The membrane voltage was stepped from Vh = −60 mV to voltages ranging between −200 and +80 mV in 40-mV increments as indicated. ΔF signals were lowpass filtered at 70 Hz (note that in these traces the relaxations are significantly distorted by the filter). (B) Na+ dependency of the voltage-dependent fluorescence (ΔF). Steady-state fluorescence at different membrane potentials was acquired for each Na+ concentration indicated on the figure. Data points are joined for visualization only. (C) Data in B were replotted as a function of the Na+ concentration and fitted with Eq. 4 (solid lines). (D) KmNa as reported by the fit of Eq. 4 to the data in C. For the initial fit we obtained H = 1.8 ± 0.02, and then refit the data with H constrained to 1.8 to reduce the fitting error associated with KmNa. (E) Li+ dependency of the voltage-dependent fluorescence (ΔF). Steady-state fluorescence at different membrane potentials was acquired for each Li+ concentration indicated in the figure. In addition, data were acquired with 1 mM Pi in 100 mM Li+. Symbols are joined for visualization only. (F) Data in E were replotted as a function of the Li+ concentration and fitted with Eq. 4 (solid lines). (G) KmLi as reported by the fit of Eq. 4 (H constrained to 1) to the data in F.
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Figure 5. Pi dependency of the voltage-dependent fluorescence in oocytes expressing S448C. (A) Original fluorescence trace recorded in ND100 (top) and ND100 + 1 mM Pi (bottom) solutions from an oocyte labeled with MTS-TMR. The membrane voltage was stepped from Vh = −60 mV to voltages in the range −200 to +80 mV in 40-mV increments, as indicated. Data were lowpass filtered at 500 Hz. (B) Pi dependency of the voltage-dependent fluorescence (ΔF). Steady-state fluorescence at different membrane potentials was acquired for each Pi concentration indicated in the figure. Data points are joined for visualization only. (C) Data in B were replotted as a function of the Pi concentration and fitted with Eq. 4 (solid lines). (D) \documentclass[10pt]{article}
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\begin{equation*}K_{{\mathrm{m}}}^{{\mathrm{P}}_{{\mathrm{i}}}}\end{equation*}\end{document}, as reported by the fit of Eq. 4 to the data in C (H constrained to 1).
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Figure 6. Presteady-state charge movements associated with S448C before and after labeling. (A) Representative current recordings from the same oocyte before (left) and after (middle) labeling with 1 mM MTSEA for 3 min for superfusion in 100 mM Na+ (ND100) and 0 mM Na+ (ND0). Voltage steps were applied from Vh = −60 mV to test potentials in the range −160 to +80 mV, as indicated. For comparison, currents from a noninjected oocyte (NI, right) from the same donor frog are also shown for the two superfusion conditions. (B) Voltage dependency of the main relaxation time constant (τON) as a function of the four Na+ concentrations indicated, before (left) and after (right) labeling for the S448C expressing oocyte in A. (C) Corresponding voltage dependency of the ON-charge (QON) associated with the main relaxation component plotted for four Na+ concentrations indicated before (left) and after (right) labeling. Continuous lines are fits with Eq. 2. The data were offset so that each curve superimposed at the depolarizing limit predicted for ND125, to better visualize the effect of Na+ on V0.5. Note the difference in ordinate scales for the control and +MTSEA conditions. (D) Summary of the Na+ dependency of the Boltzmann fit parameters, V0.5 (left), z (middle), and normalized Qmax (right). Data points are shown as mean ± SEM (n = 4). The linear regression lines for the V0.5 data were fit to data points for Na+ ≥ 25 mM. The Qmax data were fit the modified Hill equation (Eq. 1) with a variable offset and H constrained to 1, and yielded KmNa = 16 ± 6 mM for control and 38 ± 14 mM for +MTSEA. For z, data points are joined for visualization only.
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Figure 7. Presteady-state charge movements and fluorescence. (A) Simultaneous recordings of percent change in fluorescence (ΔF/F) for a representative oocyte in response to the voltage step protocol as shown, for superfusion in full Na+ (ND100), choline replacement (ND0), and Li+ replacement (LD100) and corresponding presteady-state currents (Ipss, black traces). For Ipss, the records were fit with a double decaying exponential and the main component (I2, assumed to be S448C-related is shown superimposed, red traces). For the ΔF/F records, the data were fit with a single growing exponential, shown superimposed on each trace (red traces). Recording bandwidth was 500 Hz for Ipss and ΔF/F. Baseline adjustment was performed for Ipss. (B) Parametric plot of ΔF/F against I2 as a function of time for the cell in A at the indicated test potentials. Dotted lines indicate deviations from linear behavior for hyperpolarizing potentials. (C) Voltage dependency of the time constants associated with the decay of I2 (ND100) and time-dependent phase of ΔF/F (ND100, LD100). Note that the ordinate scale covers two ranges. Data pooled from n = 5 cells.
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Figure 8. Modeling the transport cycle of NaPi-II. (A) Kinetic scheme for the transport cycle of NaPi-II. Transitions that are influenced by voltage are indicated by blue arrows and include transition of the empty carrier (1⇔8) and the second Na+ binding (2a⇔2b). No information is available on the internal substrate interaction transitions, but they are depicted to mirror those taking place on the external side and are indicated with gray arrows. The highest fluorescence is associated with states 1 and 8, and is highlighted with a red box. (B) Simulated ΔF in response to membrane voltage and Na+ concentration. The points are joined for visualization only. (C) Data in B were replotted as a function of [Na]. The solid lines represent the fit Eq. 4. The Hill coefficient reported by the fit was 1.8 ± 0.01, in excellent agreement with experimental data. (D) Simulated ΔF in response to membrane voltage and Li+ concentration (data points joined for visualization only). Based on experimental data, we assumed that Li+ only interacts with the first Na+ binding step (1-2a). (E) Data in D were replotted as a function of [Li] and fitted with Eq. 4. The Hill coefficient reported by the fit was 1.0. The rate constants (in s−1) used in the simulations were as follows: k12a = 500[Na]o or k12a = 200[Li]o, k2a1 = 900(Na+ data) or k2a1 = 40 (Li+ data), k2a2b = 10,000[Na]oexp(αeV/2kT), k2b2a = 200exp(−αeV/2kT), k81 = 40exp(−γeV/2kT), k18 = 400exp(γeV/2kT). α and γ are the fractions of the electrical field that are sensed by the hypothetical charge moving through the membrane electrical field for transitions 2a⇔2b and 1⇔8, respectively, and were set to α = 0.13 and γ = 0.4. We assumed sharp, symmetrical energy barriers for the voltage-dependent transitions. Differential equations describing the rate of change of state occupancies (Xn) were solved for the state occupancies, where n is the state. The state occupancies were constrained such that X8 + X1 + X2a + X2b = 1, and the change in fluorescence was given by ΔF = 1 − (X2a + X2b). For the simulations, T = 293 K, [Na]o varied from 0 to 0.125 M, [Li]o varied from 0 to 0.1 M.
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