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Figure A1. Simulations of slippage mode behavior. (A) Four state kinetic scheme used to simulate the slippage mode. (B) Simulated currents in response to voltage steps indicated in the inset. The parameters for the simulations on the left were: k012 = 8, 000 M−1 s−1, k021 = 2,000 s−1, k023 = 5 s−1, k014 = 120 s−1, k041 = 60 s−1, k034 = 100 s−1, k043 = 1,000 M−1 s−1, α′ = 0.3, α″ = 0.3, δ = 0.4, Nao = 0.1 M, Nai = 0.01 M, T = 20°C. The effect of alkylation (right) was simulated by a 10-fold increase in k014 and k041. (C) Expanded view of the traces in A, which also shows the steady state current levels under the two conditions. The broken line indicates zero baseline current. Note that the current scale (eu, electronic units) gives the apparent charge movement per cotransporter.
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Figure 1. Topological representation of the rat type IIa sodium phosphate cotransporter (rat NaPi-IIa). Scheme is based on current hydropathy data and a recent topology study (Lambert et al. 1999) in which eight membrane spanning regions (TM1–TM8) are predicted with extracellular loops between TM1–TM2, TM3–TM4, TM5–TM6, and TM7–TM8, and both the carboxy and amino termini are intracellular. (•) Positions of 15 residues that were individually mutated to cysteines (see methods and materials).
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Figure 4. Steady state kinetics of oocytes expressing S460C. (A) Pi dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s) at 100 mM Na+. Data points are pooled from four oocytes from the same batch. was fit to the dose–response data for each cell and the data points were normalized to the predicted maximum current. Continuous line is refit of to the pooled data, giving a Hill coefficient, nPi = 1.04 ± 0.07 and apparent Pi affinity, KmPi = 0.081 ± 0.01 mM. (B) Na+ dose response determined from original records such as shown in the inset (scale: vertical, 50 nA; horizontal, 10 s), at 1 mM Pi. Data points are pooled from three oocytes from the same batch. Data were treated as in A. Fit of (continuous line) gave a Hill coefficient nNa = 2.35 ± 0.21 and apparent Na+ affinity KmNa = 56.3 ± 4.0 mM. (C) Effect of PFA on the slippage mode (left) and cotransport mode (right) for WT (filled bars) (n = 9) and S460C (open bars) (n = 5). Inset shows an original recording from a cell expressing S460C: (1) response to 0.3 mM Pi, (2) response to 0.3 mM Pi and 3 mM PFA, (3) response to 3 mM PFA. Traces have been aligned to the baseline current in the absence of substrate (dashed line). For the slippage mode assay, bars represent the ratio of trace 3 response to trace 1 response. For cotransport mode assay, bars represent the ratio of trace 2 response to trace 1 response, both relative to the level in the presence of PFA alone (trace 3).
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Figure 2. Alkylation using MTSEA leads to a suppression of the electrogenic response in oocytes expressing mutant S460C. (A) Comparison of the Pi-induced current for an oocyte expressing the WT NaPi-IIa before and after exposure to 100 μM MTSEA (left) and two oocytes expressing S460C before and after exposure to 100 μM MTSEA or MTSET for 2 min (right). Bars represent time of application of 1 mM Pi. Dashed line indicates baseline holding current level. Note that Pi induces an upward deflection in the holding current after alkylation. (B) Restoration of Pi response by 15-min incubation in 10 mM DTT (right) for a cell expressing S460C. Incubation in 1.5 μM MTSEA exposure (middle) inhibited the initial Pi response (left) by 80%. (C) Dependency of the Pi response on MTSEA concentration. Inset gives a set of original records showing Pi response (applied during bar) for an oocyte expressing S460C after exposure to successive 10-fold increasing doses of MTSEA from 0.001 to 10 μM. Scale: vertical 50 nA, horizontal 20 s. For this cell, after exposure at 10 μM MTSEA, Pi induced an upward deflection of the baseline current. There was no change in the response after exposure to 100 μM MTSEA. Dose response is pooled from five oocytes. The Pi-induced response was calculated relative to the Pi-induced current after alkylation in 100 μM MTSEA, and was then normalized to the initial response for each cell. Curve is a fit of to the data, which gave an apparent half-maximal concentration = 0.5 μM.
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Figure 3. Immunodetection of WT and S460C protein. (A) Western blot obtained from a pool of five oocytes injected either with water, WT, or S460C cRNA. 10 μl of the lysates was separated on a 9% SDS gel and, after blotting, immunoreactive proteins were visualized by incubation with an antibody against the rat NaPi IIa NH2 terminus. This blot confirms that lysate from oocytes expressing S460C presents a similar band (97 kD) to the WT. (B) Streptavidin precipitation of oocyte lysate obtained from pools of five oocytes injected either with water, wild type, or S460C cRNA and incubated with 100 μM MTSEA-Biotin. Cells were then homogenized in 100 μl buffer (see materials and methods) and 90 μl of each lysate was incubated with Streptavidin beads. After washing, bound proteins were eluted with loading buffer (see materials and methods) and the elute was then treated as for A. Single band at 97 kD confirms that the alkylated protein was S460C.
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Figure 5. Mutant S460C and wild type show comparable voltage dependence. (A) Steady state current–voltage curves for representative cells expressing wild type (□) and S460C (▵) for the cotransport (left) and slippage (right) modes. For cotransport mode, the current (Ip) represents difference between current induced by 1 mM Pi and current in absence of Pi normalized to the value at −100 mV (n = 4). For slippage mode, the current (Is) represents difference between the holding current and current induced by 3 mM PFA, normalized to the value at −100 mV (n = 4). SEMs smaller than symbol size are not shown. (B) Pre–steady state relaxations induced by voltage steps from −60 mV holding potential to voltages in the range −140 to +80 mV in ND100 solution. Inset shows original records before (left) and after (right) application of 3 mM PFA. Q–V curve found by integrating the transient current after subtraction of the PFA response. (▪) ON charge movement, (□) OFF charge movement. Continuous line is fit of to mean of ON and OFF charges, which gave fit parameters: Qmax = 5.7 nC, z = 0.7; V0.5 = −51 mV.
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Figure 6. Effect of alkylation by MTSEA on Pi and PFA response for cells expressing S460C. Substrate was applied during the period indicated by the bar. Cell was voltage clamped to −50 mV. Note that after alkylation, the 3 mM Pi and 3 mM PFA responses superimpose and are identical to the PFA response before alkylation. Vh = −50 mV.
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Figure 7. Pre–steady state charge kinetics are faster after MTSEA incubation. Recordings of pre–steady state relaxations from a representative oocyte expressing S460C, superfused in ND100 solution (A) and ND0 solution (B) before MTSEA application (100 μM, 2 min, top) and after MTSEA application (middle). Bottom traces were recorded from a noninjected oocyte from the same batch under the same superfusion conditions (without MTSEA). In each case, oocytes were voltage clamped at −60 mV holding potential and records are shown for voltage steps according to the protocol in A. Each record is the average of eight sweeps with records obtained in 3 mM PFA, 100 mM Na+ was subtracted to eliminate capacitive charging transient. Records were low-pass filtered at 2 kHz and sampled at 50 μs/point. All records were blanked for the first 1.5 ms during the charging period of the oocyte. For this cell, the response to 3 mM Pi before MTSEA was −100 nA, and after MTSEA it was +15 nA relative to the holding current at −50 mV.
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Figure 8. Membrane potential protects against MTSEA suppression of Pi response only in the absence of external Na+. (A) Original recordings from two representative oocytes (top and bottom) from the same donor frog expressing S460C before and after a 2-min exposure to 10 μM MTSEA at different holding potentials. After alkylation, the response was tested each time with 1 mM Pi at −50 mV in ND100. (Top, 1) Initial response, (2) response after alkylation at +20 mV holding potential in ND100. (Bottom, 1) Initial response, (2) response after alkylation at +20 mV in ND0, (3) response after alkylation at −50 mV in ND0, (4) response after alkylation at −50 mV in ND100. The dashed line represents the initial holding current level before Pi application. (B) Pooled data of the inhibition of the Pi response after alkylation in 0 mM Na+, at three holding potentials. n = 3 (−20 mV, 0 mV); n = 5 (+20 mV). For all cells, after reexposure to MTSEA at −50 mV in ND100 solution, the Pi response was the same as the PFA response. The percent change in the Pi-induced electrogenic response was expressed as: 100 · [1 − (Ip+ + Is)/(Ip− + Is)], where Ip− and Ip+ are the Pi-induced current before and after MTSEA exposure, respectively, Is is the PFA-induced change in holding current (slippage current), with all currents expressed as magnitudes. It was assumed that the slippage was fully suppressed by 3 mM PFA so that the true Pi-induced response for saturating Pi was given by the change in current relative to the holding current during PFA exposure.
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Figure 9. Kinetic Scheme for Type II Na+/Pi Cotransport. States occupied in the cis (outward facing) orientation are 1–5. States occupied in the trans (inward facing) orientation are 6–10. The three modes of operation that involve transmembrane reorientation, as revealed by steady state and pre–steady state studies of WT, are indicated by dark shading: empty carrier (10 ⇔ 1), slippage (2 ⇔ 9), and cotransport (5 ⇔ 6). At least two voltage-dependent transitions have been identified: empty carrier (10 ⇔ 1) and first external Na+ binding/release step (1 ⇔ 2). The voltage dependence of the last Na+ binding/release step on the trans side (9 ⇔ 10) has not been characterized. PFA binding places the system in state 2*, which when occupied prevents the slippage and cotransport modes. The lightly shaded region indicates those transitions and associated states that have been shown to remain intact after alkylation. In addition, the zero voltage rate constants for the empty carrier mode increase after alkylation (see text).
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