Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes

Abstract

1. The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium-coupled inorganic phosphate (Na+-Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+-Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2. In this study, PKC-induced inhibition of type II Na+-Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi-activated current. 3. Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+-Pi cotransporters lost > 50 % of Pi-activated transport function when exposed to the PKC activators DOG (1,2-dioctanoyl-sn-glycerol) or PMA (phorbol 12-myristate 13-acetate). DOG-induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4alpha-PDD (4alpha-phorbol 12,13-didecanoate) showed no significant loss of cotransporter function. 4. Oocytes expressing the rat renal Na+-SO42- cotransporter alone, or coexpressing this with the type IIa rat renal Na+-Pi cotransporter, showed no downregulation of SO42--activated cotransport activity by DOG. 5. Steady-state and presteady-state voltage-dependent kinetics of type II Na+-Pi cotransporter function were unaffected by DOG. 6. DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC-mediated endocytosis. 7. Immunocytochemical studies showed a redistribution of type II Na+-Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG-induced internalization of the transport protein. 8. These findings document a specific retrieval of exogenous type II Na+-Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte.

Figure 1. Two PKC activators inhibit P_i-activated currents in oocytes expressing the type II Na⁺-P_i cotransporter NaPi-IIa/rat

A, P_i-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 P_i for 20 s. Note that the records have been truncated during the recovery phase, and in each case after removal of P_i the holding current eventually returned to the value prior to the application of P_i. For the DOG and control traces, the two oocytes were from the same donor frog. B, the time dependency of inhibition of the P_i-activated response, normalized to the peak amplitude of the initial response, resulting from incubation in 5 μm DOG (▪) (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 P_i-activated response. Twelve oocytes expressing NaPi-IIa/rat were divided into three groups, tested at -50 mV with 1 mM P_i, incubated in ND96 containing the indicated substance(s), and retested 60 min later.

Figure 2. Specificity of the DOG effect with respect to electrogenic Na⁺-P_i and Na⁺-S_i cotransport

A, pooled results for three type II Na⁺-P_i cotransporter isoforms and the rat renal Na⁺-SO₄²⁻ cotransporter (NaSi-1/rat). All responses were normalized to the peak P_i- or SO₄²⁻-activated response prior to the start of incubation. □, 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 NaSi-1/rat (□) (n = 7). All cells were from the same batch and the data are plotted as the peak P_i- or SO₄²⁻-activated current; s.e.m. for the SO₄²⁻-activated current is smaller than the symbol size.

Figure 3. Steady-state and presteady-state kinetics are unaffected by DOG

A, an isochronic plot of the response to 1.0 mM P_i ($I_{\text{p}}^{1·0}$) against the response to 0.1 mM P_i ($I_{\text{p}}^{0·1}$) 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 (□) and after 60 min () exposure to DOG (n = 4). Each bar is the response to 1 mM P_i at the indicated voltage normalized to the respective response at -50 mV. Data were obtained from I-V curves such as that shown in the inset for a typical oocyte expressing NaPi-IIa/rat at the start (▪, ▴) and after 60 min incubation in DOG (5 μm) (□, ▵). Squares and triangles represent the response to 1.0 mM P_i and 0.1 mM P_i, 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 P_i, 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 (▪) and after 30 min incubation (□). The continuous curves represent the result of fitting the Boltzmann relation (see Methods) to these data. The results of the fit are given in Table 1.

Figure 4. DOG induces a decrease in membrane capacitance (C_m) for cells expressing Na⁺-P_i type IIa and type IIb cotransporters

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 (). Pooled results of non-injected oocytes from the same batch as for the respective injected oocyte, incubated under the same conditions, are also shown (□) (n = 4). The capacitance after 60 min was normalized to the value prior to incubation in each case. C, the isochronic plot of P_i-activated current (I_p) (-50 mV, 1 mM P_i) against the corresponding change in membrane capacitance (ΔC_m) relative to the initial value, for seven individual oocytes expressing NaPi-IIa/rat and incubated in DOG (5 μm) reveals a linear correlation between I_p and ΔC_m, independent of the initial level of expression, indicated by the ordinate value at ΔC_m= 0. Each symbol represents a single oocyte. Inset shows the progressive decrease with time of normalized membrane capacitance for NaPi-IIa/rat-expressing oocytes incubated in 5 μm DOG (n = 4).

Figure 5. Immunocytochemistry detects the redistribution of NaP_i-IIa/rat and NaP_i-IIb/mse protein after DOG treatment

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.

Figure 6. Immunoblotting specific for NaPi-IIa/rat indicates DOG-induced internalization of NaPi-IIa/rat protein from the oocyte membrane

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.