INTRODUCTION

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THE CORTICAL COLLECTING DUCT (CCD) plays a key role in the hormone-regulated Na reabsorption and K secretion (8, 9). There are two types of cells in the collecting duct epithelium, principal and intercalated cells, and the principal cells are responsible for Na reabsorption and K secretion (8, 9). The K secretion is a two-step process: 1) K enters the cell across the basolateral membrane via a Na-K-ATPase; 2) K is then secreted into the lumen across the apical membrane through the SK channel. Therefore, SK channels provide the major route for K exit across the apical membrane in the CCD (6, 12, 21, 22). Thus changes in channel open probability or channel number could significantly affect renal K secretion (20). The K secretion is regulated by hormones such as aldosterone and vasopressin and by acid-base balance (1, 8, 18, 19). In addition, dietary K intake plays an important role: a high-K intake stimulates whereas a low-K intake suppresses K secretion (4, 13, 18, 24). The dietary effect on the K secretion is partially achieved by changing the apical K conductance (14, 20). Several studies have demonstrated that the number of SK channels in the apical membrane increased by 100-200% in the CCDs obtained from rats on a high-K diet in comparison with tubules from rats on a normal K diet (14, 21). The effect of high-K intake on channel number is not the result of increasing the circulating level of aldosterone, because the number of SK channels does not change in CCDs isolated from animals on a low-Na diet (14), a maneuver which significantly increases the level of aldosterone. Moreover, a high-K diet does not increase transcription of SK channels, since the mRNA encoding ROMK channels, which are closely related to apical SK channels in the CCD (5, 10, 21), was the same in CCDs from animals on a high-K or a normal K diet (7). This suggests that the effect of a high-K diet on the number of SK channels is achieved by a posttranslation modulation. In the present study, we explore the role of PTK in mediating the effect of K diet on the activity of the apical SK channels in the rat CCD.

	METHODS

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Preparation of rat CCD. The experiments were carried out in the isolated CCDs from kidneys of pathogen-free Sprague-Dawley rats. The animals were obtained from Taconic (Germantown, NY) and maintained either on a high-potassium diet (10%) or a potassium-deficient diet (Harlan Teklad, Madison, WI) for 10 days before use. The weight of animals used for experiments was between 100 and 120 g. After euthanasia, the abdomen of the animal was cut open, and the two kidneys were rapidly removed. Several thin slices of the kidney (<1 mm) were cut and placed on the ice-cold Ringer solution until dissection. The dissection was carried out at room temperature, and two watchmakers forceps were used to isolate the single CCD. The tubules were placed onto a 5-mm × 5-mm cover glass coated with Cell-Tak (Becton-Dickinson, Bedford, MA), to immobilize the tubules, and then transferred to a chamber (1,000 µl) mounted on an inverted Nikon microscope. The CCDs were superfused with HEPES-buffered NaCl solution, and the temperature of the chamber was maintained at 37 ± 1°C by circulating warm water surrounding the chamber. The CCD was cut open with a sharpened micropipette to expose the apical membrane.

Patch-clamp technique. We used an Axon model 200A patch-clamp amplifier to record channel current. The current was low-pass filtered at 1 kHz using an eight-pole Bessel filter (model 902LPF; Frequency Devices, Haverhill, MA) and digitized at a sampling rate of 44 kHz using a modified Sony PCM-501ES pulse code modulator and stored on videotape (JVC-HR-J400U). For analysis, data stored on the tape were collected to an IBM-compatible Pentium computer (Gateway 2000) at a rate of 4 kHz and analyzed using the pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channel activity was defined as NP_o, a product of channel number (N) and open probability (P_o), which was calculated from data samples of 60-s duration in the steady state as follows:
where t_i is the fractional open time spent at each of the observed current levels. The slope conductance of the channel was calculated by measurement of K current at several cell membrane potentials.

Western blot. Protein samples extracted from the kidney cortex and outer medulla were separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline (TBS), rinsed, and washed with 1% milk in Tween-TBS. The cSrc antibody was purchased from Santa Cruz (Santa Cruz, CA) and was diluted at 1: 2,500. The protein concentration used for immunoblot was 100 µg. The cSrc was detected and quantitatively analyzed using fluorescence phosphorimaging.

Measurement of the activity of cSrc tyrosine kinase. Kidney extracts prepared in RIPA buffer [150 mM NaCl, 50 mM Tris (pH 7.4), 50 mM -glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM EDTA, 5 mM EGTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 2 µg/ml pepstatin] were diluted to 1 mg/ml with the buffer. The extract (300 µl) was immunoprecipitated overnight using cSrc antibody (1:200 dilution) coupled to protein A-Sepharose. The Sepharose pellet was suspended in 50 µl of 10 mM HEPES (pH 7.4). For protein tyrosine kinase (PTK) assays, the Sepharose pellet was incubated in a total volume of 30 µl containing 100 µM [³²P]ATP (1 cpm/fmol), 12 mM magnesium acetate, 2 mM MnCl₂, 0.3 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.5 mM ammonium molybdate, and 2 mM R-R-Src peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly) (3). Reactions were quenched by adding 75 mM phosphoric acid, and 15 µl of cocktail were placed on phosphocellulose paper. The amount of ³²P incorporated was assessed using a liquid scintillation counter. In addition, 10 µl of the Sepharose pellet were boiled and used for immunoblot analysis. The protein tyrosine activity was normalized in comparison to the relative kinase concentration determined by the Western analysis using fluorescence phosphorimaging technique.

Experimental solution and statistics. The pipette solution contained (in mM) 140 KCl, 1.8 MgCl₂, and 10 HEPES (pH 7.4). The bath solution for cell-attached patches was composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.8 MgCl₂, and 10 HEPES (pH 7.4). For inside-out patches, the bath solution had the same composition as those in cell-attached except that free Ca²⁺ was reduced to 100 nM. Genistein and herbimycin A were purchased from Biomol (Plymouth Meeting, PA) and chemicals were dissolved in the DMSO solution. The final concentration of DMSO was less than 0.1% and had no effect on channel activity. Partially purified preparation of active p60-Src was obtained from Upstate (Lake Placid, NY). The activity of this enzyme was confirmed using R-R-Src peptide in in vitro kinase assays.

Data are shown as means ± SE, and an unpaired Student's t-test was used to determine the significance between two groups. Statistical significance was taken as P < 0.05.

	RESULTS

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Figure 1 shows the mean NP_o of the apical SK channel per patch in cell-attached patches in CCDs obtained from animals on either a high-K or a K-deficient diet. The mean NP_o in 88 patches was 3.1 ± 0.4 in the CCD obtained from rats on a high-K diet but decreased sharply to only 0.40 ± 0.05 (n = 67) in the CCD isolated from animals on a K-deficient diet. To explore the role of PTK in regulating the apical SK channels, we examined the effect of 100 µM genistein on the activity of the SK channels. Figure 2 is a representative recording of channel activity in a cell-attached patch in the CCD obtained from a rat on a K-deficient diet. It is apparent that the inhibition of PTK increases the activity of apical SK channels. We studied the biophysical properties of the channel shown in Fig. 2, and the result is demonstrated in Fig. 3. Figure 3A is a recording in an inside-out patch with 140 mM KCl in the pipette and 5 mM KCl in the bath, showing that the channel has a high open probability (0.90) that is not voltage dependent between 20 and 40 mV. The channel has one open time (15 ms) and one closed time (0.6 ms) (Fig. 3B). A long closed state could be observed when cell membrane potential hyperpolarized. Since events were not enough to make a curve fitting, we did not include these long closed times. The current-voltage (I-V) curve yields the slope conductance of 38 pS between 20 and 40 mV (Fig. 3C). Thus we confirmed that the newly appeared channel is the SK channel. Table 1 summarizes the effect of 100 µM genistein on channel activity. Inhibition of PTK increased the channel activity in CCDs from animals on a K-deficient diet in 11 of 12 patches, and the mean increase in NP_o was 2.5 ± 0.2. In contrast, application of genistein failed to activate the SK channel in all 10 patches in CCDs obtained from high-K-adapted rats. This suggests that PTK activity may be higher in CCDs from rats on a K-deficient diet than those on a high-K diet.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Mean NPo of each patch observed in cortical collecting duct (CCD) obtained from rats on a high-K diet or a low-K diet. NPo is a product of channel number (N) and open probability (Po). *Significant difference between two groups (P < 0.01).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   A representative channel recording showing effect of 100 µM genistein on the channel activity in CCD from rats on a low-K diet. Experiments were performed in a cell-attached patch; "C" indicates the channel closed level. Top trace: time course of effect of genistein; the 3 parts of the trace indicated by 1-3 are extended, below, to show to fast time resolution.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Channel induced by genistein in Fig. 2 is recorded in the inside-out configuration. A: channel current at different voltages. B: open and closed time histograms of SK channel. C: current-voltage (I-V) curve of SK channel.

This inference is confirmed by Western blot analysis, which shows the presence of cSrc in both renal cortex and outer medulla (Fig. 4). Kidneys from rats on a K-deficient diet express a higher level of cSrc than those on a high-K diet. Figure 5A summarizes the results obtained from six experiments. The level of cSrc in the renal cortex and outer medulla increased by 261 ± 20% in rats on a K-deficient diet compared with those on a high-K diet. We also used R-R-Src peptide as substrate (3) to measure the activity of cSrc in the kidneys and found that the specific activity of cSrc obtained from rats on a K-deficient diet is significantly higher (61 ± 8%) than those on a high-K diet (Fig. 5B).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   A Western blot shows that antibody to cSrc p60 recognizes a 60-kDa band corresponding the cSrc protein in renal cortex and outer medulla (OM) from rats on both a high-K and a low-K diet.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   A: relative intensity of cSrc protein levels in kidney from high-K and low-K adapted rats. Intensity was determined using fluorescence phosphorimaging and normalized to fluorescence intensity of rats on a high-K diet. B: relative activity of cSrc in kidneys from rats on both a high-K and a low-K diet measured with R-R-Src peptides. Activity was normalized to the level in high-K diet rats. *Significant difference between two groups (P < 0.01).

To examine whether cSrc can inhibit the SK channel, we tested the effect of exogenous purified preparation of active cSrc on the SK channel in inside-out patches. The activity of this exogenous enzyme was confirmed in in vitro kinase assay by measuring the phosphorylation rate of R-R-Src peptides. Figure 6 is a representative channel recording from three experiments showing that the effect of cSrc on channel activity in the CCD from rats on a high-K diet. Addition of 1 nM exogenous cSrc failed to inhibit the SK channel in an inside-out patch within 15-20 min. This suggests that cSrc cannot inhibit the SK channel directly.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Top: channel recording showing effect of 1 nM exogenous cSrc on channel activity in an inside-out patch. Exogenous cSrc was added directly into bath solution for 15-20 min. Bottom: all-points-histogram obtained in absence of (control) or in presence of cSrc (cSrc). NPo is the same in both cases.

To further confirm that the effect of genistein is the result of inhibition of PTK, we have examined the effect of herbimycin A, an agent which is more specific inhibitor of PTK including cSrc. Figure 7 is a representative channel recording showing the effect of 1 µM herbimycin A on the SK channels in the CCD obtained from rats on a K-deficient diet. As in the case of genistein, herbimycin A stimulated the SK channels in 14 of 16 experiments in CCDs isolated from rats on a K-deficient diet: mean increase in NP_o was 2.4 ± 0.2 (Table 2). In contrast, herbimycin A had no effect on the channel activity in the CCD from high-K adapted rats in any of 15 experiments (Table 2). In addition, effects of herbimycin A can only be observed in cell-attached patches but not in excised patches (data not shown). This indicates that the effect of herbimycin A is not the result of inhibiting the membrane-bound PTK and in turn activating SK channels. That an increase in PTK activity may be partially responsible for the low channel activity observed in the CCD obtained from rats on a K-deficient diet is further supported by experiments in which channel activity was assessed in the presence of herbimycin A (Fig. 8A). Pretreatment of CCDs with 1 µM herbimycin (20-30 min) significantly increased the mean NP_o from the control value (0.40) to 2.2 ± 0.2 per patch (n = 55) in the CCD obtained from rats on a K-deficient diet. In contrast, the mean NP_o of 30 patches carried out on those on a high-K diet was 3.25 ± 0.4, which is not significantly different from the NP_o observed under the control conditions (Fig. 8B). This strongly indicates that the activity of PTK plays a key role in regulating the apical SK channel density and that an increase in the activity of PTK such as cSrc lowers the number of the apical SK channel.

View larger version (29K): [in this window] [in a new window]   Fig. 7.   A channel recording showing effect of 1 µM herbimycin A on channel activity in CCDs obtained from rats on the low-K diet. Experiment was carried out in a cell-attached patch, and pipette holding potential was 40 mV. Top trace: time course of experiments (initial part of channel current is out of scale because of saturation of the amplifier). The 2 parts of the trace indicated by 1 and 2 are extended, below, at a fast time resolution. Channel closed level is indicated by "C," and each short line indicates channel current level.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Effect of herbimycin A treatment on mean NPo per patch in CCD from rats on a K-deficient diet (A) and in CCD from high-K diet rats (B).

	DISCUSSION

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The main finding of the present investigation is that inhibition of PTK stimulates the activity of apical SK channels in CCDs from animals on a K-deficient diet. We have demonstrated that the level of cSrc, which is the most abundant and highly expressed PTK in a variety of cells, increased significantly in kidneys from rats on a K-deficient diet in comparison with the those from rats on a high-K diet. However, it is possible that cSrc is not the only isoform of PTKs responsible for regulating the number of apical K channels in the CCD. A careful study is required to determine isoforms of PTKs that are involved in regulation of the activity of SK channels. In addition, tyrosine phosphorylation is determined by both PTK and protein tyrosine phosphatases. Therefore, it is conceivable that protein tyrosine phosphatase may be also involved in regulating the activity of SK channels. Further experiments are needed to investigate the role of protein tyrosine phosphatase in mediating the effect of K intake on K secretion.

The mechanisms by which a high intake of K augments the apical K conductance and SK activity have been investigated previously. Two findings argue against direct involvement of aldosterone. First, a low-Na diet, known to stimulate aldosterone secretion, has no effect on the number of apical SK channels (14). Second, the acute administration of aldosterone does not increase the number of SK channels (14). A permissive role of aldosterone in optimizing K secretion during chronic K loading has been reported (14) and is most likely to involve stimulation of Na-K-ATPase. The present data suggest an aldosterone-independent effect of K intake on apical SK channels. Our data support the view that a decrease in PTK levels and activity in the CCD from rats on a high-K diet increases the number of apical SK channels, whereas an increase in PTK activity from rats on a K-deficient diet depletes the apical membrane of SK channels. The mechanism by which PTK inhibits SK channels is not known. Since the effect of herbimycin A/genistein occurs quite rapidly, within 20-30 min, it is thus unlikely that changes in protein synthesis are involved.

There are at least two possibilities by which PTK can regulate the activity of SK channels. First, PTK phosphorylates the SK channel and inhibits channel activity. PTK has been shown to be involved in suppressing a delayed rectifying K channel and Kv1.3 (2, 11) and in activating Ca²⁺-dependent K channels and delayed rectifier K channels in Schwann cells by direct tyrosine phosphorylation (16, 17). Although this mechanism is not supported by our observations that the addition of exogenous cSrc did not affect channel activity in inside-out patches, we cannot exclude the possibility that PTKs other than cSrc can phosphorylate the SK channels and inhibit channel activity. Alternatively, the phosphorylation of the SK channel by cSrc may require some cytosolic factors that are absent in excised patches. Therefore, we need further experiments to examine whether ROMK channels can be phosphorylated on tyrosine residue and whether channel activity is inhibited by tyrosine phosphorylation.

Another possibility is that PTK reduces the insertion of SK channels or increases the internalization of SK channels. It is of interest that cSrc has been shown to interact with several signaling agents such as dynamin and synapsin that control membrane protein traffic (5, 15) and that overexpression of cSrc enhances endocytic internalization of epidermal growth factor receptor in fibroblasts (23).

The mechanism by which PTK activity increases in tubules from animals on a K-deficient diet is not clear. It has been demonstrated that acidosis stimulates the cSrc activity in proximal tubules (25). Since K depletion induces cytosolic acidosis ,which decreases the apical K conductance in the CCD, it is possible that changes in cell pH are involved in the regulation of PTK activity. Thus a common mechanism may be responsible for reduction of apical K conductance of the CCD from animals with acidosis or on a low-K diet. We conclude that PTK is involved in mediating effects of K intake on the apical K secretory channels in the CCD and that PTK may be a member of the aldosterone-independent signaling which regulates K secretion.