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Figure 1. . (A) Topological representation of the rat type IIa Na+/Pi cotransporter comprises a backbone of eight putative membrane-spanning domains (TMD-1 to -8) and corresponding linker regions. A functionally essential cysteine bridge is formed between Cys-306 and Cys-334 in the large second extracellular linker (ECL-2) and an additional bridge between Cys-225 and either Cys-520 or Cys-597 has also been recently proposed (Kohler et al., 2003). The cluster of functionally important (MTS-accessible) sites previously identified by SCAM in the third extracellular linker (ECL-3) (Lambert et al., 2001) and two sites in the first intracellular linker (ICL-1) (Kohler et al., 2002a) are indicated (filled squares). Sites 130â140 in the putative first extracellular linker (ECL-1) and 532â538 in the predicted fourth extracellular linker (ECL-4) were mutated to cysteines in this study (gray-filled squares) (B) Comparison of the amino acid sequences for the predicted transmembrane domains flanking ECL-1 (TMD-1 and -2) and ECL-4 (TMD-7 and -8) show a high degree of homology between different isoforms of the type II Na+/Pi cotransporter family (SLC34). Bold lettered amino acids in ECL-1 and ECL-4 for the rat isoform were mutated to cysteines for the present study. Amino acids are named using the single letter code.
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Figure 2. . Identification of functionally important sites in ECL-1 and ECL-4. (A) 32Pi uptake of WT and mutants before (filled bars) and after (unfilled bars) incubation for 10 min in 1 mM MTSEA (n ⥠10 oocytes per group). (B) Remaining electrogenic activity (expressed as % of the initial response) after 3â5 min exposure to 1 mM MTSEA for n ⥠3 oocytes/construct, measured at Vh = â50 mV, ND100 ± 1 mM Pi. WT response, filled bar; mutants, open bars. ND (not determined) refers to mutants that gave an electrogenic response <â15 nA or no measurable response to 1 mM Pi in at least three batches of oocytes from different donor frogs. Insets show representative recordings from three oocytes voltage clamped to â50 mV that expressed the WT (1), G134C (2), and F137C (3), respectively, before (â) and after (+) incubation for 3 min in 1 mM MTSEA. The response of each oocyte to a 20-s application of 1 mM Pi (open bars) and 1 mM PFA (filled bars) was recorded. Dotted lines indicate peak of Pi-dependent response and holding current in ND100 to aid comparison. Asterisks indicate mutants that showed statistically significant deviations from the WT, as reported by an unpaired t test (P < 0.05), applied to the normalized data. (C) Comparison of activity after MTSEA exposure for mutants for which reliable electrophysiological data was available (ECL-1, empty squares; ECL-4, gray-filled squares; WT, filled square) normalized to control (âMTSEA) condition. Vertical error bars indicate SEM for normalized uptake data, horizontal error bars indicate SEM from data in B. Gray bar represents mean ± SEM to indicate change of electrogenic activity observed for WT-expressing oocytes after MTSEA exposure.
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Figure 3. . Accessibility of cysteine mutants in ECL-1 (A) and ECL-4 (B) by MTSEA-Biotin. Western blots show streptavidin precipitates of MTSEA-biotinylated oocytes (top) and the corresponding whole cell lysates (bottom) from oocytes that expressed the indicated mutants probed with an antibody raised against the NH2 terminus of NaPi-IIa. Biotinylation data have been combined from several assays made from different batches of oocytes. In each assay, WT and water (H2O) injected oocytes were used as negative controls, and oocytes injected with cRNA coding for the mutant S460C (Lambert et al., 1999a) were used as a positive control. The band in the range 80â100 kD confirms that the labeled protein is NaPi-IIa. White lines indicate that intervening lanes have been spliced out.
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Figure 4. . Determination of MTS-Cys reaction rates for mutants that showed a change of activity after MTS exposure. (A) Representative recording of current from an oocyte that expressed mutant F137C after successive applications of MTSET (10 μM) (light gray bars). The cumulative exposure time (min) is indicated above each test response. Test substrates Pi (1 mM, black bars) and PFA (1 mM, dark gray bars) were applied for â¼20 s. (B and C) Pi-dependent currents at Vh = â50 mV, normalized to the initial value plotted as a function of cumulative MTS reagent exposure time for selected mutants in ECL-1 (B) and ECL-4 (C). Cells were exposed to either MTSEA (filled squares) or MTSET (open squares) for the cumulative time indicated and at the concentrations given in Table I. Continuous line is a fitted single exponential function with plateau (Eq. 1) from which the reaction rate and plateau were estimated. Each data point is pooled from â¥3 cells. Broken lines indicate plateau levels (IPiâ) estimated from the fit of Eq. 1 (Table I).
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Figure 5. . Screening for deviations from WT behavior of substrate activation kinetics at â50 mV using a two-point assay. (A) Two-point assay sensitivity for Pi activation (left) and Na+ activation (right). Continuous lines are the Pi activation index (ratio of response in 100 mM Na+ to 0.1 mM Pi and 1 mM Pi) as a function of apparent affinity for Pi (KmPi) and Na+ activation index (ratio of response to 1 mM Pi in 50 mM Na+ and 100 mM Na+) as a function of apparent affinity for Na+ (KmNa), respectively. Indices were determined as a function of KmPi or KmNa using the modified Hill equation to describe the electrogenic response to Pi, IPi = IPimax (SnH)/(SnH + KmSnH), where S is the concentration of the variable substrate, KmS is the apparent affinity constant, nH is the Hill coefficient (nH = 1.0 for Pi activation, nH = 2.5 for Na+-activation; Forster et al., 1998, 1999), and IPimax is the Pi-dependent change in holding current at the saturating limit. In general, KmS and IPimax can also depend on the concentration of the invariant substrate and holding potential (Vh). Lower test concentration was chosen close to previously reported estimates for the WT apparent affinity, and upper test concentration was chosen close to the saturation concentration for Pi or maximum usable Na+ concentration, for the Pi and Na+ activation screens, respectively. For each case, the gray bar represents range of index values observed for WT-expressing oocytes (n = 9, three donor frogs). Vertical lines indicate typical KmPi for WT (0.06 mM) and KmNa for WT (50 mM) at â50 mV, previously reported (e.g., Forster et al., 1998), respectively. (B) Pi activation index (top) and Na+ activation index (bottom), where each point is mean ± SEM for â¥4 oocytes. Gray bars indicate typical range observed for WT-expressing oocytes measured under the same experimental conditions. ND, not determined; âMTS, filled squares; +MTS, empty squares.
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Figure 6. . Currentâvoltage (IâV) curves for selected mutants. (A) Mutants that showed significantly decreased electrogenic activity at â50 mV after MTSEA exposure: I136C (1), F137C (2) in ECL-1, and M533C (3) in ECL-4, before MTSEA exposure (filled squares) and after 3â5 min exposure to 1 mM MTSEA (empty squares) (n ⥠4). G134C (4) in ECL-1 showed increased electrogenic activity at â50 mV after MTSEA exposure. Each data point is the change in holding current induced by 1 mM Pi (pH 7.4, in ND100), normalized to the magnitude of IPi at â100 mV, 1 mM Pi, before MTSEA exposure. For G134C, the data were normalized to the magnitude of IPi at â100 mV after MTSEA exposure. WT steady-state IâV data for IPi (filled circles) for WT-expressing oocytes, normalized to the magnitude of IPi at â100 mV (n = 4), are superimposed. SEMs smaller than symbol size are not shown. (B) IâV data showing the respective leak current (top) and Pi-dependent current (1 mM Pi) (bottom) for a representative oocyte that expressed M533C before (filled squares) and after (empty squares) MTSEA exposure; data points joined by dotted lines. The leak was estimated from the response to superfusion in 1 mM PFA (âIPFA). The Pi-dependent currents were leak corrected by adding the respective leak values at each test potential before MTSEA exposure (filled diamonds) and after MTSEA exposure (empty diamonds); data points joined by continuous lines. (C) IâV data showing the respective leak current (top) and Pi-dependent current (1 mM Pi) (bottom) for a representative oocyte that expressed G134C before (filled squares) and after MTSEA exposure (empty squares); data points joined by dotted lines. The Pi-dependent currents were leak corrected by adding the respective leak values at each test potential before MTSEA exposure (filled diamonds) and after MTSEA exposure (empty diamonds); data points joined by continuous lines.
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Figure 7. . Cysteine engineering at sites in ECL-1 and ECL-4 induces deviations from WT voltage dependency. (A) Voltage dependency of G134C (squares), I136C (triangles), F137C (inverted triangles), and M533C (diamonds), before (filled symbols) and after (open symbols) incubation in MTSEA (1 mM, 3 min). Pi-dependent currents, corrected for leak, were normalized to the response at 0 mV. The ordinate scale represents the relative change in IPi as a function of membrane potential. Data pooled from n > 4 cells. WT data is represented by continuous line. (B) Voltage dependency index for all functional mutants given by ratio of response to 1 mM Pi at â100 mV to that at 0 mV (100 mM Na+) before (filled squares) and after (open squares) MTS treatment (1 mM MTSEA, 3 min). Continuous reference line indicates WT index. Dotted reference line indicates that G134C-MTS, S532C ± MTS, and M533C + MTS have the same index. Arrows indicate direction of voltage dependency change for selected mutants after MTS incubation.
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Figure 8. . Graphical representation of accessibility of three putative extracellular linkers based on effective second order reaction rate k* plotted on a logarithmic scale (filled squares). A larger k* indicates that the site has a greater accessibility from the external aqueous medium. Sites that were labeled with MTSEA-Biotin, for which the respective mutants showed no detectable change in activity, are indicated (empty squares). Data for ECL-1 and ECL-4 were obtained from this study (Table I); data for ECL-3 were replotted from a previous study (Lambert et al., 2001).
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