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MS 9155 Received 15 January 1999; accepted after revision 19 February 1999.
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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-PDD (4-phorbol 12,13-didecanoate) showed no significant loss of cotransporter function.
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The homeostasis of inorganic phosphate (Pi) in mammals is primarily determined by the rate of Pi reabsorption at the brush border membrane in the renal proximal tubule. This process is mediated by an electrogenic Na+-coupled cotransport system involving a specific cotransporter protein which has been designated the type II Na+-Pi cotransporter (reviewed in Murer & Biber, 1997). Pi reabsorption via this system can be modulated directly at the functional protein level by pH, extracellular ionic composition and membrane potential (Amstutz et al. 1985; Busch et al. 1994a; Forster et al. 1997, 1998), and indirectly via humoral factors, chiefly parathyroid hormone (PTH) (Kempson et al. 1995) and dietary Pi (Levi et al. 1994), which assume important physiological roles in regulating net Pi reabsorption (for review, see Murer et al. 1991; Murer & Biber, 1997). In vivo studies have suggested that PTH induces retrieval of Na+-Pi cotransporters from the brush border membrane as indicated by a loss of immunostaining specific for type II Na+-Pi cotransport from the brush border membrane (Kempson et al. 1995; Keusch et al. 1998). This behaviour has also been confirmed using the opossum kidney (OK) cell model (Pfister et al. 1997) and previous studies indicate that the PTH action is most probably mediated via the adenylate cyclase-protein kinase A (PKA) (Caverzasio et al. 1986) and/or the phospholipase C-protein kinase C (PKC) pathways (Nakai et al. 1987; Quamme et al. 1989; reviewed in Murer et al. 1991).
In agreement with the findings from PKC activation in OK cells (Nakai et al. 1987; Quamme et al. 1989) and renal proximal tubular cells in primary culture (Friedlander & Amiel, 1989), it was recently shown that type II Na+-Pi cotransporters cloned from rat and human kidney (Magnanin et al. 1993) are also inhibited by PKC activation when expressed in Xenopus laevis oocytes (Hayes et al. 1995; Wagner et al. 1996). These findings suggested that the Xenopus laevis expression system might provide a useful model for detailed characterization of type II Na+-Pi cotransporter regulation. However, in these studies no direct evidence was reported to indicate whether the PKC-induced inhibition was mediated by changes to the type II Na+-Pi cotransporter protein itself (e.g. phosphorylation) thereby modulating the cotransporter kinetics, or altered membrane trafficking which would lead to endocytotic removal of cotransporters from the membrane, as suggested from the in vivo studies.
The aim of this present study was therefore to identify and characterize the mechanism by which the transport function of type II Na+-Pi cotransporters expressed in Xenopus laevis oocytes is inhibited when exposed to membrane-permeable PKC activators. Using electrophysiological and immunodetection techniques, we show that the PKC-induced downregulation of transport function, quantified in terms of Pi-activated electrogenic currents in Xenopus oocytes, results from a decrease in the number of functional cotransporters in the membrane, rather than a modulation of cotransporter kinetics. Furthermore, we provide clear evidence that in Xenopus oocytes the PKC effect is mediated by membrane retrieval of functional type II Na+-Pi transport protein from the cell membrane and that the type II Na+-Pi transport system represents a specific target when the PKC pathway has been activated.
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METHODS |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Oocytes
All animal handling was carried out in accordance with methods approved by Swiss Government authorities. Female Xenopus laevis frogs were anaesthetized with 0·5 % methanesulfonate salt (Sigma) and placed on ice, and oocytes were removed from the ovaries and prepared according to standard procedures. Frogs were allowed to recover postoperatively before being returned to the aquarium facilities. Stage V-VI oocytes were injected (50 nl injection volume) with approximately 10 ng oocyte-1 of cRNA encoding the desired protein 24-48 h after defolliculation as previously described (Forster et al. 1998). In the case of coinjected oocytes, the injection volume per oocyte remained constant and equal volumes of the respective cRNAs were mixed prior to injection. Cells were incubated at 16-18°C in modified Barth's solution (see below) and tested for expression 2-5 days after injection. Only cells having a resting membrane potential exceeding -20 mV and a leakage current not exceeding -50 nA at -50 mV were used for the subsequent experiments.
cRNA and cotransporter nomenclature
For convenience we have used the following nomenclature for the type II Na+-Pi cotransporters used in this study: NaPi-IIa/rat (Magnanin et al. 1993), NaPi-IIb/flr (Werner et al. 1994) and NaPi-IIb/mse (Hilfiker et al. 1998), where the species are rat, flounder (flr) and mouse (mse). The subdivision of type II Na+-Pi cotransporters into types IIa and IIb is based on the classification introduced with respect to the recently cloned type II Na+-Pi cotransporter from mouse intestine (Hilfiker et al. 1998). The rat renal Na+-coupled sulfate cotransporter (Markovich et al. 1993) was designated NaSi-1/rat.
Electrophysiology and data acquisition
Oocytes were placed in a small recess in a Plexiglass superfusion chamber (volume, 0·2 ml) and superfused at 2-5 ml min-1 using gravity feed with ND96 control solution together with the indicated substance (see below). During the incubation period, between substrate test applications, superfusion was stopped to preserve solutions. After substrate application, superfusion was continued for up to 1 min to ensure return of the response to the baseline value. In some experiments, oocytes were first tested electrophysiologically, then removed from the recording chamber, placed in the incubation medium at 18°C and retested electrophysiologically after 60 min. All superfusates were cooled to 18°C before entering the recording chamber. The long-term stability of the cell was monitored using a chart recorder and each new test solution application was only made after the holding current had returned to a stable baseline.
Oocytes were voltage clamped using a custom-built two-electrode voltage clamp with active series resistance compensation to improve the clamping speed for the membrane capacitance and presteady-state measurements and to improve the accuracy of membrane potential control in the case of large steady-state Pi-activated currents.
Cells were impaled with microelectrodes pulled from borosilicate glass (Type 14 074 20, Hilgenberg, Germany) on a programmable pipette puller (BB-CH, Mecanex, Switzerland) using a three-stage pulling sequence. Pipettes were filled with 3 M KCl. Voltage electrodes had resistances in the range 3-4 M
, and current electrodes had a typical resistance of 0·5 M.
For assessing steady-state transport activity, currents were measured at a holding potential of -50 mV to reduce possible contamination from Ca2+-activated Cl- currents at depolarized potentials. The exposure time to Pi was kept to a minimum (typically 20 s) to reduce possible intracellular accumulation of substrate. Current-voltage (I-V ) curves were generated using a staircase protocol with 20 mV steps of 100 ms duration from -120 to +20 mV as described by Forster et al. (1998).
All current recordings were filtered using an 8-pole Bessel filter (Frequency Devices, Model 902 or 828, Haverhill, MA, USA) at a cut-off frequency less than twice the sampling frequency used. Data acquisition, voltage command generation and solution valve control were performed using laboratory-built PC-compatible hardware and programmed as previously described (Forster et al. 1997, 1998).
Solutions and chemicals
All standard chemicals and reagents were obtained from Sigma or Fluka (Buchs, Switzerland). Staurosporine, 1,2-dioctanoyl-sn-glycerol (DOG), phorbol 12-myristate 13-acetate (PMA) and 4-phorbol 12,13-didecanoate (4-PDD) were obtained from Sigma; bisindolylmaleimide I (BIM I) was obtained from Calbiochem. Elugent and protease inhibitor cocktail for surface biotinylation were obtained from Calbiochem and Sigma, respectively.
The composition of the solutions used was as follows. (i) Oocyte incubation solution (modified Barth's solution) (mM): NaCl, 88; KCl, 1; CaCl2, 0·41; MgSO4, 0·82; NaHCO3, 2·5; Ca(NO3)2, 0·33; and Tris, 7·5, pH 7·6; supplemented with antibiotics (10 mg l-1 penicillin-streptomycin). (ii) Control superfusate (ND96) (mM): NaCl, 96; KCl, 2; CaCl2, 1·8; MgCl2, 1; and Hepes, 5, titrated to pH 7·4 with NaOH. (iii) Substrate test superfusate: control superfusate together with either inorganic phosphate (Pi) as KH2PO4/K2HPO4, proportioned to give pH 7·4, or inorganic sulfate as Na2SO4.
PKC activators and inhibitors were all dissolved in DMSO and stored at -20°C, and were added to the test superfusate immediately before use. After dilution, the final concentration of DMSO to which the oocytes were exposed did not exceed 0·05 %.
Data analysis and curve fitting
Non-linear regression analysis was performed using Inplot version 4.0 or Prism version 2.0 software (Graphpad Inc., San Diego, CA, USA). All data are shown as means ± S.E.M. and n is the number of oocytes. Experimental protocols were repeated at least twice on different batches of oocytes from different frogs.
Membrane capacitance was determined by measuring the current transient in response to 20 ms voltage steps from -50 to -40 mV and integrating the respective on- or off-transients to give the charge transfer. Capacitance was then calculated assuming an invariant intrinsic membrane capacitance of 1 µF cm-2. No significant difference was found between the on- and off-charge, indicating that the system was linear. Based on the presteady-state charge-voltage (Q-V) data, contamination from charges associated with the cotransporters would be expected to contribute < 10 % of the total charge under these conditions.
Analysis of presteady-state relaxations was performed by fitting a single exponential to the main current relaxation, commencing approximately 4 ms after the voltage step was applied, i.e. after the charging of the oocyte capacitance was complete. A curve-fitting algorithm based on the Chebychev transformation (G. Malachowski, personal communication) was used. The apparent charge transfer was then estimated from the integral of the fitted relaxation. To characterize the Q-V data and thereby estimate the total maximum translocatable charge (Qmax), a Boltzmann equation was fitted to these data of the form:
| Q = Qhyp + Qmax/(1 + exp(-ze(V - V0·5)/kT)), | (1) |
where Qhyp, which depends on the holding potential, is the charge translocated at the hyperpolarizing limit, V0·5 is the voltage at which the charge is distributed equally between the two states, e is the electronic charge, z is the valency, k is the Boltzmann constant, and T is the absolute temperature.
Estimation of cotransporter turnover
Cotransporter turnover, 
, which in the present case reflects the rate of translocation of Pi, was estimated from steady-state and presteady-state parameters (e.g. Loo et al. 1993; Mager et al. 1993; Wadiche et al. 1995). The steady-state Pi-induced current at a holding potential, V, can be expressed as:
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Ip(V) = Ntqt ((V)),
| (2) |
where Nt is the number of cotransporters and qt is the net charge transferred per cycle, where it is assumed that qt is independent of V. For type II Na+-Pi cotransport, direct assays of the Na+ : Pi stoichiometry have shown that one net charge is transferred per cycle (Forster et al. 1999), hence qt = 1·602 × 10-19 C. Furthermore, Nt can be expressed in terms of the presteady-state parameters as:
| Nt = Qmax/ze, | (3) |
where it is tacitly assumed that all Nt cotransporters contribute equally to both the steady-state and presteady-state currents. The cotransporter turnover at any potential V is then given by:
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(V) = (Ip(V) )z/Qmax. (4)
| (4) |
Immunocytochemistry
Oocytes were fixed by immersion in a solution comprising 3 % paraformaldehyde and 0·1 % glutaraldehyde in phosphate-buffered saline (PBS) for 30 min. After rinsing with PBS at 4°C, cells were frozen onto thin cork slices using liquid N2-cooled liquid propane. Sections (5 µm) were cut at -20°C with a cryomicrotome and mounted on chromalum-gelatine-coated glass slides. For immunofluorescence staining, sections were pre-treated for 10 min with PBS containing 3 % milk powder and 0·3 % Triton X-100 and then incubated for 12 h with primary antibodies raised against the NaPi-IIa/rat (Custer et al. 1994) and NaPi-IIb/mse (Hilfiker et al. 1998) Na+-Pi cotransporters, diluted 1 : 500 in PBS containing 3 % milk powder and 0·3 % Triton X-100. Sections were then rinsed 4 times with PBS and incubated for 1 h at room temperature with pig anti-rabbit IgG-conjugated fluorescein isothiocyanate (FITC; Dakpotts, Glostrup, Denmark), diluted 1 : 50 in PBS-milk powder-Triton X-100 solution (see above). After rinsing with PBS, sections were placed on coverslips using DAKO Glycergel (Dakopatts) plus 2·5 % 1,4-diazabicyclo[2.2.2.]octane (DABCO, Sigma) as a fading retardant. Immunofluorescence was revealed by fluorescence light microscopy (Polyvar, Reichert-Jung, Austria) using narrow-band filter systems for FITC.
Membrane purification
For the detection of NaPi-IIa/rat protein in the membrane, the procedure of Turk et al. (1996) was used. Briefly, cells were homogenized (20 µl oocyte-1) by pipetting in a lysate buffer containing 100 mM NaCl, 20 mM Tris-HCl and 1 % elugent, protease inhibitor cocktail, at pH 7·6. Cells were incubated for 5 min on ice, and the homogenate was then centrifuged (15 000 g, 3 min, 4°C) and the supernatant (85 %) was retained for gel electrophoresis and immunoprecipitation. Protein determinations were made using the Bradford assay. Equivalent amounts of protein were used for Western blots (see 'Gel electrophoresis and immunoblots', below) of NaPi-IIa/rat from the cell lysate (25 µg) or for streptavidin precipitation (75 µg).
Gel electrophoresis and immunoblots
The method was similar to that described by Hayes et al. (1994). Briefly, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes. Polyclonal antibodies raised against synthetic peptides corresponding to the deduced amino-terminal amino acid sequence of the NaPi-IIa/rat protein were used to detect NaPi-IIa/rat-related immunoreactivity. Following overnight incubation with the primary antibody (1 : 2000 dilution) and incubation with a goat anti-rabbit IgG conjugated with horseradish peroxidase (1 : 1000 dilution), antibody reactivity in oocyte membranes was visualized using an enhanced chemiluminescence (ECL) detection system (Amersham). Non-specific binding of the antibody was prevented by blocking with Tris-buffered saline (TBS; 0·9 % NaCl, 10 mM Tris-HCl, pH 7·4) containing 5 % non-fat milk powder in 1 % Triton X-100 and washing as previously described (Pfister et al. 1997).
Cell surface biotinylation
Biotinylation of surface-expressed protein was performed using the procedure described by Hayes et al. (1994). Oocytes were washed 5 times for 5 min in Barth's solution (see above) containing 10 mM triethylamine, pH 9·0 at 4°C, and then incubated in the same mixture supplemented with 2·2 mM of the biotinylation reagent sulfosuccinimidobiotin long chain (1 : 200 dilution from frozen aliquots in DMSO) (NHS-LC-biotin, Pierce) at 4°C. The solution was replaced after 30 min and the oocytes incubated for a further 30 min at 4°C in the presence of the biotinylation reagent. The labelling solution was then replaced with Barth's triethylamine solution containing 5 mM glycine for 10 min, and oocytes were washed twice for 5 min in Barth's triethylamine solution. The yolk-free homogenates were prepared as described above (see 'Membrane purification').
Streptavidin precipitation
The supernatant of the oocyte lysate was incubated (2 h with end-over-end rotation at 4°C) with 40 µl streptavidin-agarose beads, prewashed twice in lysate buffer. The beads were collected and washed 3 times in lysate buffer. Bound proteins were dissociated from the beads with 2 × Laemmeli buffer (4 % SDS, 40 % glycerol, 240 mM Tris-HCl, 100 mM dithiothreitol, pH 6·8) at 95°C for 3 min followed by brief centrifugation. All samples were stored at -80°C. Biotinylated NaPi-IIa/rat-related proteins were detected by gel electrophoresis and immunodetection as described above.
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RESULTS |
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PKC activators inhibit the steady-state Pi-activated current
We first confirmed a previous finding that prolonged exposure to phorbol esters, known to activate protein kinase C (PKC), led to a progressive inhibition of the Pi-activated current (Ip) recorded under voltage clamp conditions from Xenopus oocytes expressing cloned renal type II Na+-Pi cotransporters (Hayes et al. 1995; Wagner et al. 1996). In the present case, a 67 ± 8 % (n = 4) inhibition of Ip was observed in oocytes expressing the rat renal Na+-Pi cotransporter (NaPi-IIa/rat), voltage clamped to a holding potential (Vh) of -50 mV and incubated with 50 nM phorbol 12-myristate 13-acetate (PMA) for 60 min. Figure 1A (upper traces) shows a typical recording in which the inhibition was 77 %. As a negative control, we also confirmed that 60 min incubation with the inactive phorbol ester 4-phorbol 12,13-didecanoate (4-PDD) (50 nM) did not result in significant inhibition of Ip (n = 2) (data not shown).
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A, Pi-activated responses of oocytes expressing NaPi-IIa/rat before (left-hand traces) and after 60 min incubation (right-hand traces) in ND96 with the indicated substance: upper traces, 50 nM PMA; middle traces, 5 µM DOG; lower traces, control solution (ND96). The cell was continuously voltage clamped at -50 mV and activity was tested with 1 mM Pi for 20 s. Note that the records have been truncated during the recovery phase, and in each case after removal of Pi the holding current eventually returned to the value prior to the application of Pi. For the DOG and control traces, the two oocytes were from the same donor frog. B, the time dependency of inhibition of the Pi-activated response, normalized to the peak amplitude of the initial response, resulting from incubation in 5 µM DOG ( | ||
For the remainder of this study we used 1,2-dioctanoyl-sn-glycerol (DOG) for the purpose of PKC activation because of its higher potency in activating PKC and the absence of artifactual effects on oocytes reported in the case of phorbol esters (Hirsch et al. 1996; J. Hirsch, personal communication). Figure 1A (middle traces) shows typical steady-state Pi-activated currents recorded from another oocyte expressing NaPi-IIa/rat at a Vh of -50 mV in ND96 medium containing 5 µM DOG. The peak Ip decreased to approximately 25 % of the original response, which was comparable with the PMA data. For the control oocyte from the same donor frog, which was incubated in ND96 solution without DOG (lower traces), a 12 % rundown of Ip was observed over the same incubation interval.
The inhibition of Ip by DOG developed progressively with time as shown in Fig. 1B with data pooled from NaPi-IIa/rat-expressing oocytes from three different donor frogs (see inset for original tracings from a typical oocyte). Typically there was 50 % inhibition of Ip after 30 min incubation, followed by a more gradual decline towards a plateau after 60 min. For some batches of oocytes, the time course was found to vary such that no significant change was observed in Ip until after 30 min. In all cases, the apparent rundown observed for oocytes not exposed to DOG never exceeded 20 % of the initial response (Fig. 1B) and, moreover, there was no significant effect on Ip of exposure to the vehicle, DMSO (0·05 %), alone (data not shown).
To provide additional confirmation that the inhibition of Ip by DOG was due to the activation of intracellular PKC, we co-incubated oocytes with DOG (5 µM) alone and DOG (5 µM) together with one of two commonly used PKC inhibitors: staurosporine (5 µM) and bisindolylmaleimide I (BID I) (5 µM). In this experiment, all oocytes were from the same batch and were tested consecutively. For each oocyte, Ip was measured before and after a 60 min exposure. As shown in Fig. 1C, in the presence of each inhibitor, after 60 min incubation, there was only partial suppression of the Pi-induced response compared with DOG alone. This finding indicated that the DOG-induced inhibition of Ip was specifically due to activation of the PKC pathway. Incubation for 60 min with the inhibitors alone did not result in a significant change (< 10 %) in the initial Ip measured (data not shown).
Cotransporter-specific action of DOG
Next we studied the specificity of the DOG effect on cotransporter function. As summarized in Fig. 2A, in contrast to the 
30 % inhibition exhibited by this batch of oocytes expressing the NaPi-IIa/rat cotransporter, oocytes expressing the electrogenic renal Na+-coupled sulfate transporter (NaSi-1/rat) (Busch et al. 1994b) showed no significant change in the electrogenic response to 5 mM Na2SO4. Moreover, both oocytes expressing the NaPi-IIb/flr isoform and those expressing the NaPi-IIb/mse isoform showed similar inhibition of Ip when exposed for 60 min to DOG to that of oocytes expressing the NaPi-IIa/rat isoform.
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A, pooled results for three type II Na+-Pi cotransporter isoforms and the rat renal Na+-SO42- cotransporter (NaSi-1/rat). All responses were normalized to the peak Pi- or SO42--activated response prior to the start of incubation. | ||
To investigate this effect further and test the hypothesis that the presence of Na+-Pi type II protein per se might be necessary for inhibition of the NSi-1/rat response, we coexpressed NaPi-IIa/rat and NaSi-1/rat in oocytes and measured the response to 1 mM Pi and 5 mM Na2SO4 consecutively at 10 min intervals. As shown in Fig. 2B, whereas the response to Pi showed the same time course of inhibition as observed in oocytes expressing NaPi-IIa/rat alone, the response to Na2SO4 showed a significant increase (80 %, n = 7) after 10 min exposure and then decreased again to the level prior to exposure to DOG. It should be noted that no measurable electrogenic responses were observed in oocytes expressing NaPi-IIa/rat alone in response to Na2SO4 or in oocytes expressing NaSi-1/rat alone in response to Pi (data not shown). This confirmed that electrogenic responses for co-expressed oocytes were indeed substrate and protein specific.
These findings suggested that DOG-induced inhibition of Ip involved a downregulation mechanism common to the different type II Na+-Pi cotransporter isoforms which, however, was inactive in the case of NaSi-1/rat.
Kinetics and turnover of type II Na+-Pi cotransport are unaffected by DOG
One candidate hypothesis that could account for the decrease in Ip induced by exposure to DOG could be that the apparent affinity for Pi is progressively decreased due to modification of the type II Na+-Pi cotransporter protein and thereby effects a change in kinetics. To test this hypothesis we exploited the time-dependent inhibition of Ip (Fig. 1B) by successively applying two Pi concentrations at 10 min intervals: 0·1 mM, close to the apparent Km for Pi (KmPi), and 1 mM, to evoke a response close to saturation as previously established for NaPi-IIa/rat (Busch et al. 1994a; Forster et al. 1997, 1998). If there were a shift in KmPi during the incubation in DOG, we would predict that a plot of the current activated by 1 mM Pi (Ip1·0) against that activated by 0·1 mM Pi (Ip0·1) at each test time would deviate from a straight line through the origin. The resulting isochronic plot of Ip1·0 vs. Ip0·1 shown Fig. 3A, for three representative oocytes expressing NaPi-IIa/rat from the same donor frog, shows a clear linear correlation passing through the origin which would be the behaviour predicted for an invariant KmPi.
A second candidate hypothesis to account for the DOG-induced diminished apparent transport rate relates to the intrinsic voltage dependence of type II Na+-Pi cotransport in the steady state. All measurements so far were performed at -50 mV and since the apparent substrate affinities are voltage dependent, changes in these kinetics alone could significantly affect the transport rate (Busch et al. 1994a; Forster et al. 1997, 1998). Therefore we investigated whether the decrease in Pi-activated current was reflected in a change of the steady-state current-voltage (I-V ) relationship. I-V curves were obtained during the steady-state plateau region using a fast voltage staircase protocol (see Methods) at 0·1 and 1·0 mM Pi as shown in Fig. 3B (inset) for a typical oocyte expressing NaPi-IIa/rat. Each data point represents the difference between the response with Pi and in the absence of Pi. The I-V curves at 0 and 60 min show similar voltage-dependent behaviour before and after exposure to DOG for the two Pi concentrations used. This is illustrated in Fig. 3B with I-V data for 1 mM Pi pooled from four oocytes, in which the response over the voltage range -100 to -20 mV, normalized to the respective Ip at -50 mV, is compared at 0 min and after 60 min exposure to DOG. The similarity of the two data sets confirmed the lack of an effect of DOG on the steady-state voltage dependence despite a 76 % inhibition of the absolute responses (-50 mV, 1·0 mM Pi). Similar behaviour was obtained with 0·1 mM Pi (data not shown).
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A, an isochronic plot of the response to 1·0 mM Pi (Ip1·0) against the response to 0·1 mM Pi (Ip0·1) for three oocytes expressing NaPi-IIa/rat and incubated in 5 µM DOG. Measurements were made at 10-15 min intervals and each symbol represents data from one cell. The straight line is a linear regression line forced through the origin. B, the pooled steady-state I-V data before ( | ||
A third candidate kinetic parameter, the modulation of which could account for the DOG effect, is the cotransporter turnover rate. Based on macroscopic steady-state current measurements alone, it is not possible to distinguish between a change in the number of cotransporters contributing to Ip and a change in the turnover rate of the cotransporter itself, both of which could influence the observed macroscopic current. Although turnover is a microscopic property, it can be estimated from macroscopic measurements of steady-state current and presteady-state charge movements (see eqn (2), Methods).
In the presence of 100 mM Na+ and the absence of saturating Pi, oocytes expressing type II Na+-Pi cotransporters exhibited readily resolvable presteady-state relaxations. Figure 3C (left-hand records) shows a typical family of such relaxations for voltage steps from a holding potential of -100 mV to test potentials in the range -140 to +80 mV for a representative oocyte expressing NaPi-IIa/rat. Exposure of this oocyte to 5 µM DOG led to a significant reduction in the presteady-state currents after 45 min (Fig. 3C, right-hand records). To quantify the apparent charge movements associated with these relaxations, we fitted single exponentials to the off-transitions before and after 30 min exposure to DOG, at which time it was still possible to resolve the Na+-Pi type IIa-related relaxations from the endogenous membrane charging. The mean time constant for the off-relaxations to -100 mV from initial potentials from -140 to +80 mV did not change significantly after DOG treatment (7·44 ± 0·1 vs. 7·46 ± 0·1 ms at 30 min). For this experiment, the off-relaxations were fitted because we found that the smaller amplitude relaxations at 30 min precluded reliable fitting of the on-transitions over the whole voltage range. This result also provided additional evidence that the voltage dependence of the type II Na+-Pi cotransporter was unaffected by DOG. Figure 3D shows the apparent charge transfer associated with the relaxations plotted as a function of voltage for the oocyte before and after 30 min incubation in DOG. By fitting a single Boltzmann function (see eqn (1), Methods) to these data, we obtained estimates for the apparent valency (z), mid-point voltage (V0·5) and maximum charge movement (Qmax). Table 1 summarizes these data for this representative cell. Whereas Qmax and Ip at -50 mV (Ip(-50)) (at 1 mM Pi) decreased significantly, the change in z and V0·5 was not significant based on the error reported for fitting eqn (1). This result indicated that the voltage dependence of presteady-state charge movements, in agreement with the finding for the steady-state voltage dependence, was most probably unaffected by DOG. The transporter turnover at -50 mV ((-50)) was then estimated using eqn (4) (see Methods) and as indicated in Table 1, after 30 min exposure to DOG, (-50) did not change significantly.
Table 1. The effect of 30 min DOG exposure on steady-state and presteady-state parameters for a representative oocyte expressing NaPi-IIa/rat protein
| Time (min) |
Ip(-50) (nA) |
Qmax (nC) |
V0·5 (mV) |
z | (-50) |
| 0 | 120 | 5·1 | -37·8 | 0·66 | 15·5 |
| 30 | 66 | 2·5 | -31·3 | 0·59 | 15·6 |
(-50), Ip and at a Vh of -50 mV.
Inhibition of Ip correlates with a decrease in membrane capacitance
The above findings strongly suggested that the observed inhibition of the Pi response through DOG activation of the PKC pathway resulted from a reduction in the number of active transporters in the membrane. However, based on this evidence alone, we were unable to conclude whether DOG-induced cotransporter retrieval per se occurred, or whether the protein simply remained in the membrane in an inactivated conformation. Since membrane retrieval is implicitly associated with endocytosis of membrane vesicles and a concomitant change in membrane area, as reported for other cotransport proteins (e.g. Vasilets et al. 1990; Hirsch et al. 1996), we investigated next whether changes in membrane area were also detectable after DOG incubation for the type II Na+-Pi cotransport system. To test this hypothesis, we quantified the membrane area in terms of the membrane capacitance (Cm) obtained from the integral of the transient current in response to small voltage steps (see Methods). Figure 4A shows typical capacitive transients recorded before and after 60 min exposure to DOG for an oocyte expressing NaPi-IIa/rat. These traces indicated that a clear decrease in the oocyte charging current occurred which, in this case, was equivalent to a decrease in Cm from 177 to 96 nF, with a concomitant decrease in Ip (-50 mV, 1 mM Pi) from -112 to -38 nA. We observed a decrease in Cm for all three type II Na+-Pi cotransporter isoforms (Fig. 4B). No apparent changes in the shape or size of the oocytes were observed under these conditions. Moreover, to ascertain whether this effect was due to the presence of exogenous protein or simply an intrinsic property of the oocyte, we determined Cm for non-injected oocytes from the same respective batches of oocytes as those expressing type II Na+-Pi cotransporters. As shown in Fig. 4B, there was no significant change in Cm after 60 min exposure to DOG. To take account of the variability of this parameter between cells, the data were normalized to the Cm of each cell before exposure to DOG and then pooled. We also investigated whether incubation in PMA (50 nM) rather than DOG would lead to a reduction in Cm of non-injected oocytes, as previously reported (Vasilets et al. 1990). In three out of four batches of cells tested, there was a consistent decrease of the endogenous Cm which, however, did not exceed 11 % of the initial value (data not shown) after 60 min exposure.
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A, capacitive transients recorded from a representative oocyte expressing NaPi-IIa/rat recorded before (thin trace) and after 60 min (thick trace) incubation in DOG (5 µM). Traces were recorded in response to a voltage jump from -50 to -40 mV. The integral of each trace gives a measure of the membrane area. B, pooled results from one representative oocyte from batches expressing NaPi-IIa/rat, NaPi-IIb/flr and NaPi-IIb/mse ( | ||
From these findings we concluded that DOG incubation led to both a reduction in type II Na+-Pi cotransport function and a concomitant reduction in cell membrane area. Moreover, this latter effect was significantly greater than any change in Cm occurring in non-injected oocytes induced by either the phorbol ester PMA or DOG. This suggested that the presence of type II Na+-Pi cotransporter protein in the membrane was a necessary condition for membrane area reduction.
Like the time dependence of Ip (Fig. 1B), Cm for oocytes expressing type II Na+-Pi cotransporters also decreased progressively during continuous exposure to DOG and typically approached a plateau after 40-50 min as shown for the NaPi-IIa/rat isoform (n = 4) (see Fig. 4C inset). Comparable results were obtained for oocytes expressing NaPi-IIb/mse (data not shown). Moreover, since both the Pi-activated current and membrane capacitance showed a time dependence with exposure to DOG, we examined whether there was a correlation between these two parameters by plotting Ip isochronically against the corresponding change in Cm (
Cm) for individual cells expressing NaPi-IIa/rat, as shown in Fig. 4C. These data suggested a linear correlation between Ip and Cm. When a linear regression was performed, the mean slope was 0·63 ± 0·05 nA nF-1 (n = 7). This parameter was independent of the initial level of expression which varied approximately threefold, based on the initial values of Ip. For NaPi-IIb/mse, a similar correlation was obtained (data not shown), giving a mean slope of 0·57 ± 0·06 nA nF-1 (n = 7). The slopes for these two isoforms were not significantly different (Student's unpaired t test, P < 0·05) and their similarity suggested that the decrease in transport function and membrane area was mediated by a mechanism common to these two type II Na+-Pi cotransporters.
Immunodetection indicates internalization of Na+-Pi cotransporters from the oocyte membrane
The above findings, based on electrophysiological measurements, were compatible with the hypothesis that activation of the oocyte PKC pathway involved retrieval of functional type II Na+-Pi cotransporters from the oocyte membrane. Additional confirmation of this hypothesis was provided by immunocytochemistry.
We fixed oocytes which had previously been tested for transport function both with and without DOG treatment and prepared them for immunocytochemistry (see Methods). As shown in Fig. 5A and B, respectively, oocytes expressing NaPi-IIa/rat and NaPi-IIb/mse, when assayed using antiserum directed against a synthetic peptide corresponding to the N-terminus of the respective proteins, gave a bright fluorescence immunostaining localized in the oolemma. In contrast, two different oocytes, from the same respective batches as in Fig. 5A and B, no longer showed fluorescence confined to the oolemma after 60 min incubation in DOG. For the injected oocytes, DOG treatment resulted in the fluorescence being widely distributed in the intracellular space immediately below the plasma membrane (Fig. 5C, NaPi-IIa/rat; Fig. 5D, NaPi-IIb/mse). For these oocytes, the Pi-activated currents at the time of fixation were inhibited by > 60 % in both cases.
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Cryosections of paraformaldehyde-fixed oocytes were assayed with antiserum directed against a synthetic peptide corresponding to the N-terminus of the NaPi-IIa/rat (A and C) or NaPi-IIb/mse (B and D) protein. Specific immunostaining appears as bright fluorescence. In each case the staining of a representative oocyte without DOG treatment is shown in the upper panels: the distribution of NaPi-IIa/rat or NaPi-IIb/mse protein is confined to the oolemma. Oocytes from the same respective batches as those in A and B, after DOG treatment (1 h, 5 µM), show a clear redistribution of protein staining below the oolemma with a broad region extending 25 µm below the surface (C and D). All cells had previously been tested electrophysiologically for adequate expression of the respective cotransporter. Magnification, × 400. | ||
As an additional confirmation of internalization, we biotinylated surface-expressed protein and using immunoblotting we tested specifically for the presence of biotin-labelled NaPi-IIa/rat protein in the membrane after streptavidin precipitation (Fig. 6B). For the untreated cells a band corresponding in molecular weight to that obtained in the Western blot analysis (Fig. 6A) indicated that NaPi-IIa/rat was still present in the membrane after biotinylation and streptavidin precipitation, whereas cells which had been preincubated (60 min) with DOG and then biotin labelled showed no evidence of labelled NaPi-IIa/rat in the membrane. For the particular batch of oocytes used in this experiment, electrophysiological confirmation of the downregulation by DOG indicated that even after 30 min incubation in DOG, < 10 % of transport function remained, which was in qualitative agreement with the absence of biotin-labelled NaPi-IIa/rat in the membrane. Moreover, the Western blots of total NaPi-IIa/rat protein in oocytes showed the expected strong band at 80-90 kDa with or without DOG treatment, which indicated that this did not lead to degradation of functional protein.
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A, Western blots of oocytes incubated in control ND96 solution with (+) and without (-) 5 µM DOG. For the latter, a clear band is revealed in the 80-90 kDa range as expected for the NaPi-IIa/rat isoform (Custer et al. 1994). This band remained after DOG treatment, which indicated that the NaPi-IIa/rat protein was not degraded. B, oocytes were surface labelled with biotin following incubation in control ND96 solution with (+) and without (-) 5 µM DOG. For oocytes expressing NaPi-IIa/rat, surface labelling followed by streptavidin precipitation and immunoblotting specific for NaPi-IIa/rat protein revealed a band in the 80-90 kDa range corresponding to the Western blot result and thereby confirming the presence of the protein in the membrane. For the DOG-treated cells, no band was detected, indicating the disappearance of NaPi-IIa/rat from the membrane. Non-injected oocytes, subjected to the same incubation conditions and handling, were used as a negative control in A and B. Representative cells from the same batches used here and injected with NaPi-IIa/rat cRNA were tested for adequate expression and downregulation by DOG. | ||
Taken together, these immunodetection findings provided independent evidence that DOG induced membrane retrieval of functional type II Na+-Pi cotransporters and, furthermore, they indicated that the protein was not in a degraded form in the intracellular compartment.
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Does PKC activation alter the kinetics or number of type II Na+-Pi cotransporters?
In this study we have demonstrated that activation of the PKC pathway in Xenopus oocytes expressing three type II Na+-Pi cotransporter isoforms led to a significant inhibition of Pi-activated currents. These currents were used as a measure of transport function and an indirect measure of the number of Na+-Pi transporter proteins in the membrane. A central question concerns the identification of the mode of action of PKC in regulating the transport function. This could be the result of changes to the cotransporter protein (e.g. direct post-translational modification) by, for example, phosphorylation or interaction with some modifying factor, leading to altered kinetics or inactivation. Alternatively, membrane trafficking of type II Na+-Pi cotransporters could be altered, leading to a reduction in the total number of proteins in the membrane.
As a first approach to studying PKC-induced inhibition of type II Na+-Pi cotransporters, Hayes et al. (1995) removed five putative concensus sites for potential PKC-mediated phosphorylation to produce a NaPi-IIa/rat polymutant. Although this protein exhibited steady-state transport kinetics (Pi and Na+ activation) similar to the wild-type (WT) NaPi-IIa/rat, oocytes expressing either the WT or polymutant protein showed similar inhibition of steady-state Pi activation at -50 mV when incubated in 4-PDD. A similar finding was reported in the case of the cloned GABA transporter GAT1, where the removal of three consensus PKC sites did not affect modulation by PMA or BIM (Corey et al. 1994). These findings suggested that non-canonical PKC phosphorylation sites might exist or that the type II Na+-Pi cotransporter protein is not phosphorylated by PKC. Alternatively, PKC activation could lead to phosphorylation of regulatory proteins associated with membrane trafficking of type II Na+-Pi protein. As yet a membrane trafficking role for one recently identified candidate gene product associated with Na+-Pi type IIa cotransporters has not been established (Custer et al. 1997).
In the present study, we employed two approaches to investigate the effect of PKC activation on the type II Na+-Pi cotransport system: immunodetection and electrophysiology. These two approaches provide complementary information regarding the fate of type II Na+-Pi cotransporters and, taken together, they provide evidence to suggest that the inhibition observed can be accounted for by membrane retrieval of type II Na+-Pi cotransporter alone. The most obvious evidence for membrane retrieval was obtained from two immunodetection assays: after PKC activation, there was a redistribution of fluorescence-labelled protein from the oolemma to intracellular compartments; and after PKC activation, NaPi-IIa/rat protein was not detectable as biotinylated surface protein. However, these techniques are only semi-quantitative and, moreover, they do not provide information regarding the dynamics of the retrieval process, or address the possibility that the kinetics of the cotransporter could also be modulated via activation of the PKC pathway.
Electrophysiological techniques, on the other hand, provide an indirect, but 'real-time' probe for the effect of PKC activation on the cotransporter itself and, in particular, alterations in kinetics. Yet, on their own, the results obtained cannot unequivocally indicate membrane retrieval, since cotransporters could remain in the membrane in an inactivated conformation which would be undetectable by electrophysiological means. In the present study, we have demonstrated, based on analysis of both the steady-state and presteady-state kinetics of the NaPi-IIa/rat protein, that activation of the PKC pathway does not lead to altered kinetics. In addition to an invariance of the Pi-activation kinetics and steady-state voltage dependence, the behaviour of the presteady-state relaxations offered a further insight into the downregulation process.
Presteady-state relaxations are a general feature of electrogenic cotransporters and the currents most probably represent intramembrane movement of a fixed number of charges per cotransporter molecule (e.g. Loo et al. 1993). The magnitude of the relaxations should therefore provide a relative measure of the number of activatable cotransporters in the membrane, even if the exact charge per cotransporter is unknown. Moreover, freeze-fracture studies on oocytes indicate a direct correlation between Qmax and the intramembranous particle density in the case of the sodium-dependent glucose transporter SGLT-1 (Zamphigi et al. 1995; Wright et al. 1997). Our finding that Qmax decreased with DOG incubation, without a significant change in the voltage-dependent presteady-state kinetics, is therefore consistent with a decrease in the number of membrane-bound and activatable type II Na+-Pi cotransporter proteins.
How specific is PKC action on cotransporters expressed in oocytes?
The consistency of our results with those of other studies involving the regulation of exogenously expressed cotransporters in Xenopus oocytes (e.g. Na+-glucose (SGLT-1), Hirsch et al. 1996; taurine (TAUT), Loo et al. 1996; dopamine, Zhu et al. 1997; and GABA (GAT1), Corey et al. 1994), might suggest that the PKC-induced membrane retrieval and consequent loss of transport function are simply a general feature of activation of the oocyte regulatory pathways.
Although we cannot fully exclude non-specific effects, we provide two lines of evidence that strongly suggest that in Xenopus oocytes type II Na+-Pi cotransporters are specifically targetted for membrane retrieval. First, in non-injected oocytes, significant changes in membrane area, as indicated by capacitance measurements, were not observed after incubation in either DOG or PMA for periods of up to 60 min. This observation contrasts with the findings of Vasilets et al. (1990), who reported that treatment of non-injected oocytes with PMA, at the same concentration used in the present case, led to a significant decrease in the endogenous Na+-K+-ATPase activity and a concomitantly large decrease (66 %) in the oocyte capacitance. In our hands we were unable to observe decreases in Cm exceeding 20 % in non-injected oocytes, whereas oocytes from the same respective batches expressing Na+-Pi type IIa and type IIb isoforms consistently showed both inhibition of cotransport function and a decrease in capacitance induced by either DOG or PMA. Our findings also accord with other studies (J. Hirsch, personal communication) with respect to the regulation of SGLT-1 induced by DOG (Hirsch et al. 1996; Wright et al. 1997).
Second, whereas the Pi-activated response for three type II Na+-Pi cotransporter isoforms was inhibited, under the same incubation conditions, the SO42--activated response of the Na+-SO42- cotransporter remained essentially constant. This result also agreed with the findings of Wagner et al. (1996), using PDD as a PKC activator. We have further demonstrated the specificity of this regulation by coexpression of NaPi-IIa/rat with NaSi-1/rat. Here, an increase of the NaSi-1/rat response during the period of maximum decrease of NaPi-IIa/rat function suggested that PKC first induces an upregulation of NaSi-1/rat followed by a return to the initial level. Although we did not investigate the time course of the NaSi-1/rat activity further, it was clear that in contrast to the effect on NaSi-1/rat, PKC activation resulted in a significant downregulation of NaPi-IIa/rat. Such specificity of action has been reported in the case of primary cultures of renal proximal tubular cells, where PKC activation led to a downregulation of the Na+-dependent Pi and hexose uptake, but not of the Na+-dependent alanine uptake (Friedlander & Amiel, 1989). Moreover, in vivo studies of rat renal proximal tubules have documented no disappearance of Na+-SO42- cotransporters accompanying internalization of type II Na+-Pi cotransporters following PTH stimulation (M. Lötscher, J. Biber & H. Murer, unpublished observations).
Quantifying the retrieval of type II Na+-Pi cotransport protein
The correlation between Pi-activated current and change in membrane capacitance for NaPi-IIa/rat and NaPi-IIb/mse isoforms indicated a constant decrease in the number of functional transporters retrieved per change in membrane area. Moreover, this behaviour was independent of the absolute level of expression, which suggested that the removal of membrane is coupled to the number of cotransporters being retrieved, as would be expected if endocytosed vesicles were loaded with a fixed number of proteins. Such a correlation between steady-state activation and change in membrane capacitance has previously been observed for the rabbit isoform of the Na+-glucose cotransporter SGLT-1 induced by PKC and PKA activators (Hirsch et al. 1996; Wright et al. 1997), the insertion of Na+ pumps stimulated by botulinum C3 ADP-ribosyltransferase (Schmalzing et al. 1995) and Na+ channels (Isom et al. 1995). Moreover, there is strong evidence from morphological studies to support the conclusion that changes in membrane capacitance do indeed reflect changes in membrane area and, in particular, the density and length of microvilli (Isom et al. 1995; see also Vasilets et al. 1990; Hirsch et al. 1996).
This retrieval process can be quantified using parameters derived from the presteady-state and steady-state analyses. Since, for a constant turnover and net inward charge movement per cycle, the number of functional cotransporters is proportional to Ip (eqn (2)) and the membrane area can be related to Cm, assuming a constant intrinsic capacitance (Cm° = 1 µF cm-2), the change in the number of functional cotransporters per change in membrane area can be expressed as:
Nt/A =  Cm°/qt,
| (5) |
where is the slope of the Ip(-50) vs. Cm plot, is the cotransporter turnover (see eqn (4)) and qt is the net charge cotransported. Using the values from Table 1 in eqn (5) gives Nt/A = 2·7 × 103 cotransporters µm-2. If the process of membrane retrieval involves the formation of vesicles having a typical diameter of 120 nm (e.g. Wright et al. 1997), we estimate that approximately 120 cotransporters would be retrieved per vesicle, in agreement with the estimate of 100-200 SGLT-1 proteins per vesicle made by Hirsch et al. (1996), also based on electrophysiological measurements. Such a protein density per vesicle would most probably be impossible from the viewpoint of protein packing, assuming NaPi-IIa/rat has similar dimensions to other proteins of comparable molecular weight (e.g. Schneider et al. 1998). Indeed, the electron microscopy and freeze-fracture studies reported by Wright et al. (1997) suggest a more realistic density of only 20 SGLT-1 molecules per vesicle. To reconcile this discrepancy, Hirsch et al. (1996) proposed that the rate of exocytosis, in the case of upregulated SGLT-1, must therefore exceed the net rate of vesicle insertion since Cm only reflects the net change in membrane area. In addition to sequestration of retrieved protein to another subcellular compartment, as evidenced by immunocytochemistry findings, our data could also support a scheme involving recycling of empty vesicles: the overall net decrease in Cm observed would indicate that PKC activation involves a differential alteration in the rate of vesicle retrieval and insertion.
However, it must be noted that the apparent valency (z) determined from fitting a single Boltzmann function to the presteady-state Q-V data would underestimate the total charge per cotransporter if the protein comprised identical subunits, each contributing a charge ze (Zamphigi et al. 1995). Moreover, the application of a single Boltzmann process is most probably an oversimplification based on detailed studies of the presteady-state kinetics of the type II Na+-Pi system (Forster et al. 1997, 1998). In either case, it follows that underestimating z would lead to a smaller turnover (eqn (4)) and a concomitantly larger estimate for Nt/A (eqn (5)). For example, if the type II Na+-Pi cotransporter were assembled as a tetramer, as suggested by radiation inactivation studies (Xiao et al. 1997), comprising four functional homomonomers, Nt/A would decrease fourfold, and by setting z = 0·6, a more feasible estimate of about 30 transporters retrieved per vesicle would be obtained.
Conclusions
In this study we have combined two complementary approaches to investigate the effect of activating the PKC pathway on the type II Na+-Pi cotransport system expressed in Xenopus oocytes. The essential finding is that type II Na+-Pi cotransporters are specifically regulated by a PKC-dependent pathway, which induces protein retrieval from the oocyte membrane. That this is in accord with the behaviour of the same cotransport system in in vivo studies on the proximal tubule and in in vitro studies using the OK cell model, would support the use of the Xenopus laevis expression system for further studies. These would include identification of structural requirements at the level of the transporter molecule and additional mechanisms involved in the retrieval process.
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This work was supported financially by grants to H. M. from the Swiss National Science Foundation (SNF: 31-46523) the Hartmann Müller-Stiftung (Zurich), the Olgar Mayenfisch-Stiftung (Zurich) and the Schweizerischer Bankgesellschaft (Zurich) (Bu 704/7-1).
Corresponding author
I. C. Forster: Physiologisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Email: forster{at}physiol.unizh.ch
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) (n = 8). The normalized response from oocytes incubated in control medium (ND96) only is also shown (
) (n = 3). Data were pooled from oocytes from three different donor frogs and one control oocyte was taken from each batch. Inset shows representative traces recorded at 10 min intervals during DOG incubation. Note that as for the pooled data, DOG induced a large decrease in the response between 20 and 30 min. Test conditions as in A. C, effect of two PKC inhibitors, bisindolylmaleimide I (BIM I, 5 µM) and staurosporine (STAU, 5 µM) on DOG (5 µM) inhibition of the Pi-activated response. Twelve oocytes expressing NaPi-IIa/rat were divided into three groups, tested at -50 mV with 1 mM Pi, incubated in ND96 containing the indicated substance(s), and retested 60 min later.
, a typical control oocyte from the same respective batch, incubated in ND96 alone for 60 min; 
, cells expressing the indicated RNA incubated in ND96 and 5 µM DOG for 60 min. The intrinsic rundown was always significantly less than the DOG-induced inhibition. The number of cells in each group is indicated in parentheses. Test conditions as in Fig. 1. B, the effect of DOG (5 µM) incubation on oocytes coexpressing NaPi-IIa/rat (
) and after 60 min incubation in DOG (5 µM) (
). Squares and triangles represent the response to 1·0 mM Pi and 0·1 mM Pi, respectively. Points have been joined by lines for visualization only. C, presteady-state relaxations recorded from an oocyte expressing NaPi-IIa/rat, bathed in ND96 in the absence of Pi, before (left-hand records) and after (right-hand records) 45 min incubation in DOG (5 µM). Voltage steps (indicated in inset) were from a holding potential of -100 mV. Each trace is the mean of four records. Filtering was at 500 Hz. The endogenous charging transients of the oocyte have been truncated. The decrease in steady-state level reflects an improvement in the oocyte leakage current for this particular cell over the measurement period and this effect has also been seen in cells not exposed to DOG. D, the presteady-state charge (Q) as a function of membrane voltage (V) for the same cell as in C, prior to DOG incubation (