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Figure 1. Pi‐induced NaPi‐IIa and NaPi‐IIb currents in Xenopus oocytes. (A) schematic representation of the puff application system for TEVC recording. The oocytes were placed just under the tip of the puff pipette. B–C, traces of representative currents generated by mNaPi‐IIa (B) and mNaPi‐IIb (C) expressed in Xenopus oocytes. Oocytes were bathed in Ca2+‐free ND100 solution and clamped at ‐60 mV. Thick bars show the period of application of 10 mmol/L Pi solution.
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Figure 2. Ci‐VSP‐induced PI(4,5)P2 depletion has no effect on NaPi‐IIa but inhibits KCNQ2/3 activity. (A) pulse protocol (upper) and membrane currents (lower) in an oocyte coexpressing mNaPi‐IIa, rKCNQ2, rKCNQ3, and Ci‐VSP. rKCNQ2 and rKCNQ3 form heterotetrameric rKCNQ2/3 channels, which carry delayed‐rectifier voltage‐gated potassium currents sensitive to PI(4,5)P2. To activate Ci‐VSP, oocytes were depolarized to +50 mV for 10 sec twice. The first 10‐sec depolarization was applied before application of Pi in the bath solution and the second after application of Pi. To measure rKCNQ2/3 currents, depolarization steps to −10 mV were applied for 200 msec, 5 sec before and 5, 25, and 55 sec after each 10‐sec depolarization. The inset shows rKCNQ2/3 currents recorded to monitor the PI(4,5)P2 levels on an enlarged timescale at times 1–4 and 5–8 of the pulse protocol. Membrane potential was maintained at −60 mV throughout the recording. (B) mNaPi‐IIa current trace shown on an enlarged current scale indicated by the double arrow in A. (C) changes in mNaPi‐IIa (open squares and solid line) and rKCNQ2/3 (filled circles and dashed line) current amplitudes in response to PI(4,5)P2 depletion induced by Ci‐VSP activation. Left axis shows the scale for the mNaPi‐IIa current and the right axis shows the scale for the rKCNQ2/3 current. (D) summary of the effect of Ci‐VSP‐induced PI(4,5)P2 depletion on mNaPi‐IIa and rKCNQ2/3 currents (n = 4). Current amplitudes after the 10‐sec depolarization were normalized to the amplitudes before depolarization. Data are means ± SEM.
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Figure 3. Effect of Ci‐VSP‐induced PI(4,5)P2 depletion on NaPi‐IIb activity. (A) Pulse protocol (upper) and membrane currents (lower) in an oocyte coexpressing mNaPi‐IIb, rKCNQ2, rKCNQ3, and WT Ci‐VSP. The protocol is the same as in Fig. 2A. The inset shows rKCNQ2/3 currents recorded to monitor the PI(4,5)P2 levels on an enlarged timescale at times 1–4 and 5–8 of the pulse protocol. (B) mNaPi‐IIb current trace on an enlarged current scale shown by the double arrow in A. An arrow points to the current decrease after the first 10‐sec depolarization in the absence of Pi. (C) Representative mNaPi‐IIb current trace on an enlarged current scale obtained from an oocyte coexpressing with rKCNQ2, rKCNQ3, and C363S Ci‐VSP. The protocol is the same as in A. (D) Changes in the amplitudes of currents generated by mNaPi‐IIb in cells coexpressed WT Ci‐VSP (open squares and solid line) or C363S Ci‐VSP (filled circles and dashed line). (E) Summary of the effect of Ci‐VSP‐induced PI(4,5)P2 depletion on mNaPi‐IIb currents. Current amplitude after the 10‐sec depolarization was normalized to that before depolarization. Normalized current amplitudes were compared using Student's t‐test (n = 3 for both WT and C363S Ci‐VSP, means ± SEM, **P < 0.01).
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Figure 4. Pi‐induced NaPi‐IIa and NaPi‐IIb currents in Neuro 2a cells. (A) Representative current trace recorded from an untransfected Neuro 2a cell. Neuro 2a cells exhibit no endogenous current in response to application of 5 mmol/L Pi solution. Cells were bathed in 140 mmol/L NaCl solution. (B) Representative current traces recorded from an untransfected (left) and a mNaPi‐IIa‐expressing (right) Neuro 2a cell bathed in 140 mmol/L NaCl solution. Na+‐free solution was applied during the period indicated by the thick bar. (C) Representative current trace recorded from a mNaPi‐IIa‐expressing Neuro 2a cell. An inward current was elicited upon application of 5 mmol/L Pi solution. (D) Left: representative time course of SCN5A (hH1) current decline upon rapid perfusion of Na+‐free solution by using an ALA Scientific perfusion system. Right: current traces recorded from a HEK293T cell expressing SCN5A (hH1) (upper) and the pulse protocol (lower). Eight current traces recorded at different times during the solution exchange are superimposed. Currents were induced by 10‐msec step pulses to ‐20 mV every 1 sec after starting perfusion at 0 sec; the pulse was repeated eight times. The holding potential was −80 mV. (E) Representative current trace recorded from a mNaPi‐IIb‐expressing Neuro 2a cell. 5 mmol/L Pi solution was applied during the period indicated by the thick bar. An arrow points to the current decay. (F) Relative amplitudes of currents recorded 19 sec after reaching its maximal amplitude. Currents were normalized to the maximal amplitude. Normalized amplitudes were compared using Student's t‐test (means ± SEM, ***P < 0.001). (G) The trace in E shown on an enlarged timescale (gray trace). The current decay was fitted by a single‐exponential equation (red line). The time constant is the mean ± SEM (n = 8). In A, B, C, and E, membrane potential was clamped to −60 mV.
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Figure 5. Changes in plasma membrane PI(4,5)P2 and PI(4)P upon activation of PJ in Neuro 2a cells. (A) schematic representation of PI(4,5)P2 and PI(4)P depletion upon PJ recruitment to the plasma membrane. PI(4,5)P2 and PI(4)P were monitored using PHPLC
δ1‐mCherry and GFP‐P4M‐SidMx1, respectively. After PI(4,5)P2 or PI(4)P depletion by PJ, these fluorescent probes translocate to the cytoplasmic region. Sac 1 and INPP5E are 4‐phosphatase and 5‐phosphatase, respectively. INPP5E, inositol polyphosphate‐5‐phosphatase E; PI, phosphatidylinositol. (B) Fluorescence images of PHPLC
δ1‐mCherry before (left) and after (right) application of rapamycin (upper). Representative time course of normalized PHPLC
δ1‐mCherry fluorescence intensity in the plasma membrane and cytoplasmic region (lower). Images were captured at 0 sec (left) and 118 sec (right) from a Neuro 2a cell expressing PHPLC
δ1‐mCherry, PJ, and Lyn11‐FRB. Scale bar, 5.0 μm. Rapamycin (10 μmol/L) was applied to the recording chamber at the time indicated by the arrow in the lower panel. (C) Fluorescence images of GFP‐P4M‐SidMx1 (upper) and representative time course of normalized GFP‐P4M‐SidMx1 fluorescence intensity in each region (lower). Neuro 2a cells expressing GFP‐P4M‐SidMx1, PJ, and Lyn11‐FRB were studied. Images were captured at 0 sec (left) and 118 sec (right). Scale bar, 5.0 μm. Rapamycin was applied at the time indicted by the arrow in the lower panel.
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Figure 6. Effect of PJ‐induced depletion of PI(4,5)P2 and PI(4)P on NaPi‐IIa and NaPi‐IIb activities in Neuro 2a cells. (A) Schematic representation of the measurement of Na‐Pi cotransporter activity with PJ‐induced depletion of PI(4,5)P2 and PI(4)P. Rapamycin links PJ to Lyn11‐FRB present in the plasma membrane. (B) Representative current trace recorded from a Neuro 2a cell coexpressing mNaPi‐IIa with PJ and Lyn11‐FRB. Rapamycin (1 μmol/L) was applied after Pi‐induced transporter currents emerged. (C) Representative mNaPi‐IIb currents recorded from Neuro 2a cells coexpressing PJ and Lyn11‐FRB in the presence (upper) or absence (lower) of 1 μmol/L rapamycin. In B and C, membrane potential was clamped at −60 mV and dashed lines indicate the initial current level before perfusing Pi‐containing solution. (D) Comparison of the amplitudes of mNaPi‐IIa currents recorded before (pre) and after (post) perfusion of rapamycin. (E) Changes in mNaPi‐IIb current amplitude elicited by perfusion of rapamycin (open squares and solid line) or vehicle (filled circles and dashed line). (F) Summary of the effect of PJ‐induced depletion of PI(4,5)P2 and PI(4)P on mNaPi‐IIa and mNaPi‐IIb currents. Current amplitudes after rapamycin application were normalized to the amplitudes before application. Normalized amplitudes were compared using Student's t‐test (n = 6 for mNaPi‐IIa, n = 7 for rapamycin‐treated mNaPi‐IIb, n = 3 for vehicle‐treated mNaPi‐IIb, means ± SEM, **P < 0.01).
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Figure 7. Conceptual model showing the different PI(4,5)P2 dependences of the electrogenic NaPi‐IIa and NaPi‐IIb cotransporters. NaPi‐IIa and NaPi‐IIb transport one HPO
4
2− with three Na+. NaPi‐IIb activity, for example in the intestine, is regulated by PI(4,5)P2 in the plasma membrane. NaPi‐IIa activity in the kidney is unaffected by PI(4,5)P2.
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