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Figure 1. . Secondary topology of NaPi-IIa showing the eight putative transmembrane domains based on hydrophobicity analysis and topology studies. Two N-glycosylation sites in the second extracellular loop are indicated by hexagons and a cysteine bridge has been identified between Cys-306 and Cys-334. Squares indicate sites where cysteine substitution and/or exposure to MTS reagents alter transport function. The boxed residues in ECL-3 represent a region that forms a proposed 2.5 turn α-helix (Lambert et al., 2001). The bold regions of ICL-1 and ECL-3 represent two stretches of â¼50 amino acids that show high similarity (Kohler et al., 2002).
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Figure 2. . Effect of MTS reagents on electrogenic response of C-C mutant. (A) Representative current recordings from oocytes that expressed the single mutants A203C, S460C, and WT NaPi-IIa, before and after incubation in 100 μM MTSEA, MTSES, or MTSET (data shown only for MTSEA as MTSET and MTSES gave similar records). Traces show responses of oocytes to 1 mM Pi (filled bar) and 3 mM PFA (open bar) applied for the period indicated, before (â, left traces) and after (+, right traces) incubation. The continuous line indicates the holding current level reached during the initial PFA application. Dashed line represents holding current (Ihold) in ND100 solution. Note that for each cell, the recording baseline levels were not adjusted before and after MTSEA exposure. Note the quantification of transport modes as indicated: substrate-induced currents (IPFA, IPi) are measured relative to Ihold. Uniport or leak activity is then given by âIPFA and the cotransport activity is given by IPi â IPFA. We assume that these substrate concentrations are sufficient to inhibit fully the leak current and that both transport modes are mutually exclusive. Bars: vertical, 50 nA; horizontal, 20 s. (B) Recordings from oocytes expressing the double mutant A203C-S460C (C-C) before (â, left traces) and after (+, right traces) incubation for 3 min with 100 μM MTSEA, MTSES, or MTSES. Note that for the double mutant, the leak current increases only in the presence of MTSEA and MTSES. Bars: vertical, 50 nA; horizontal, 20 s. (C) Pooled data for the leak for oocytes expressing WT, A203C, S460C, and C-C mutant after incubation with MTSEA (white bars), MTSES (black bars), or MTSET (gray bars). Each data point represents mean of five oocytes, normalized to the value before MTS exposure.
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Figure 3. . Substrate dose responses for C-C mutant and WT before and after modification with MTSEA. (A) Pi activation for WT (open diamonds) and C-C mutant (filled squares) before modification with MTSEA at Vh = â50 mV, measured in ND100 with Pi as variable substrate. For the C-C mutant, data are also shown after incubation with MTSEA (open squares), by measuring the change in holding current induced by Pi relative to the holding current in 0 mM Pi. Each data point represents mean ± SEM (n = 4). The modified Hill equation was fit to the data to give: KmPi = 0.06 ± 0.010 (C-C, âMTSEA); KmPi = 0.06 ± 0.01 (C-C, +MTSEA) and KmPi = 0.060 ± 0.01 (WT), nH was constrained to 1.0 for fit. (B) Na+ activation for WT (open diamonds) and C-C mutant (filled squares) before modification with MTSEA at Vh = â50 mV measured at 1 mM Pi with Na+ as variable substrate. For the C-C mutant, data are also shown after incubation with MTSEA (open squares) by measuring the change in holding current induced by Pi relative to the current in 0 mM Pi. Data were pooled as in A (n = 4); fit of Hill equation gave: KmNa = 52 ± 8 (C-C, âMTSEA), KmNa = 55 ± 6 (C-C, +MTSEA), KmNa = 61 ± 10 (WT), nH = 2.3, 2.7, and 2.1, respectively. Points without error bars have SEMs smaller than the symbol size.
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Figure 4. . Characterization of other double mutants. (A) 32Pi uptake of cells injected with WT and mutant cRNA and water-injected oocytes. The bars represent the mean ± SEM (n = 8). Only the WT, C-C, S-C, C-A, and C-L mutants gave statistically significant 32Pi uptake compared with control oocytes (Students t test, P < 0.05). (B) Protein expression of mutants in oocytes. Western blot of whole cell lysate from a pool of 5 oocytes injected with cRNA coding for the indicated mutants as well as the wild-type (WT) and water-injected oocytes as controls. NaPi-IIa was visualized using an antibody raised against the rat NaPi-IIa -NH2 terminus. (C) Surface biotinylation of mutant constructs. Western blot obtained from a pool of eight oocytes injected either with water, WT or the indicated mutants cRNA. 5 μl of whole oocyte lysate (left) or 15 μl of streptavidin-precipitate of MTSEA-biotinylated oocytes expressing the indicated mutants or WT and water injected oocytes (right). Band at 93 kD confirms that the labeled protein is NaPi-IIa. (D) Immunocytochemical detection of WT NaPi-IIa and C-C mutant and β-actin in cryosections of Xenopus oocytes. Oocytes injected with H2O, or cRNA for WT, or C-C mutant were cut and cryosections labeled with a rabbit polyclonal antibodies directed against the NH2 terminus of NaPi-IIa and with phalloidin-Texas red. Specific immunostaining appears in the oocyte surface as indicated by the actin signal. No specific NaPi-IIa staining was seen for H2O injected oocytes. Magnification, 60Ã. (E) Representative electrogenic response to 1 mM Pi and 3 mM PFA for double mutants. Traces show Pi and PFA-induced currents obtained from oocytes expressing the mutated constructs, measured at 1 mM Pi (filled bar) or 3 mM PFA (open bar) in ND100 and Vh = â50 mV. Test substrates were applied for 20 s during period indicated by bars.
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Figure 5. . Effect of MTS reagents on electrogenic response of functional double mutants with single novel Cys. Representative current recordings from oocytes that expressed S-C and C-A mutants before (â, left traces) and after (+, right traces) incubation for 3 min with 10 μM MTSEA, MTSES, or MTSET (data shown for MTSEA). Each oocyte was tested with 1 mM Pi (filled bar) and 3 mM PFA (open bar) for the period indicated. The continuous line indicates level reached during the initial PFA application. Dashed line represents baseline current in ND100 solution. Note that for each cell, the recording baseline levels were not adjusted before and after MTSEA exposure.
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Figure 6. . Quantification of cysteine modification reaction in terms of cotransport loss and leak gain. (A) Loss of cotransport function (filled symbols) and gain in leak (open symbols) for C-C mutant. Oocytes expressing the mutant were incubated for the indicated cumulative time with a fixed concentration (5 μM) of MTSEA (squares) or MTSES (triangles) and the electrogenic response to 1 mM Pi (cotransport mode) and 3 mM PFA (leak mode) was measured. The transport activity was normalized as explained in the text. Continuous (MTSEA) and dotted (MTSES) lines are fits to the data with a single exponential function of the form: exp(â[MTS] t k), for normalized cotransport loss, or: 1 â exp(â[MTS] t k), for normalized fractional leak gain, where [MTS] is the concentration of MTS reagent (in μM), t is the cumulated exposure time (s) and k is the effective second order rate constant (μMâ1 sâ1). Each data point represents mean ± SEM (n = 6). Points without error bars have SEMs smaller than symbol size. (B) Isochronic plot of normalized cotransport loss and fractional, normalized leak gain for the same cells as in A, for the MTSEA exposure. Each symbol represents a different oocyte.
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Figure 7. . Interpreting the effect of MTS reagents on the transport properties of NaPi-IIa, based on an alternating access kinetic scheme. (A) Before Cys modification, transport activity assumes either leak mode (left) or cotransport mode (right), depending on the availability and concentration of external Pi. It is assumed that the internal concentrations of Na+ and Pi are such that the cycle proceeds in the direction indicated. (B) After Cys-modification, leak mode still operates (left) in the absence of external Pi. In the case of MTSES/MTSEA, a higher turnover rate is also predicted that could result from modification of rates associated with the transition 2â7. For external Pi > 0, the probability of occupancy of state 3 increases, thereby reducing the probability of transition 2â7 occurring, so that the leak activity is progressively suppressed in a dose dependent manner as the concentrations of external Pi and/or Na+ increase (see text). At saturating Pi, and Na+, the probability of occupying state 4 is high, but the translocation transition (4â5) is prevented by the modified Cys residue.
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