INTRODUCTION

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THE APICAL K CHANNELS in the medullary thick ascending limb (mTAL) play an important role in K recycling across the apical membrane of the mTAL (6, 8, 27). K recycling is essential for maintaining the function of the Na-K-Cl cotransporter, and inhibition of the apical K channels diminishes the transport rate of epithelial NaCl in the mTAL (6, 8). We have previously demonstrated that the open probability of the apical K channels in the mTAL was significantly lower in animals on a K-deficient (KD) diet than that in animals on a high-K (HK) diet (9). It is conceivable that a decrease in the activity of the apical K channels is partially responsible for hypokalemia-induced impairing of the transepithelial transport in the mTAL (10, 19, 23). Although the mechanism by which K depletion reduces the activity of the apical K channels is not completely understood, several factors have been suggested to be responsible for the effect of K depletion on the apical K channels in the mTAL. For instance, it was demonstrated that K depletion significantly decreased the expression of ROMK channels in the cell membrane of the renal medulla (16). We have also reported that 20-hydroxyeicosatetraenoic acid (HETE) production increased in the mTAL harvested from rats on a KD diet and that inhibition of cytochrome P-450-dependent 20-HETE production significantly increased the activity of the 70-pS K channel in the mTAL from rats on a KD diet (9). This suggests that a posttranslational modulation of channel activity is also involved in downregulating the activity of the apical K channels.

Furthermore, earlier investigation found that the expression and activity of c-Src increased significantly in the renal cortex and medulla in rats on a KD diet (28). Moreover, inhibition of protein tyrosine kinase (PTK) with herbimycin A significantly stimulated the low-conductance K channel in the cortical collecting duct (CCD) from rats on a KD diet as well as the ROMK1 channel in Xenopus oocytes coexpressed with c-Src (17, 28). In the present study, we examined the role of PTK and protein tyrosine phosphatase (PTP) in regulating the apical K channels in the mTAL. We discovered that stimulation of tyrosine phosphorylation decreases, whereas stimulation of tyrosine dephosphorylation increases, the activity of the apical 70-pS K channel but has no effect on the 30-pS K channel in the mTAL.

	METHODS

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Preparation of the TAL. Pathogen-free Sprague-Dawley rats (50-60 g, either sex) were purchased from Taconic (Germantown, NY). The animals were pair fed with either a HK diet (10% wt/wt) or a KD diet (<0.001% wt/wt) (Harlan, Teklad, Madison, WI) for 10 days before use. The plasma Na and K concentrations in animals on a different K diet were measured with flame photometry (Corning 480), and the results are included in Table 1. The rats were killed by cervical dislocation after anesthesia with Metofane. The kidneys were removed immediately, and thin coronal sections were cut with a razor blade. We used only medullary TALs in the study. The dissection buffer solution contained (in mM) 140 NaCl, 5 KCl, 1.8 MgCl₂, 1.8 CaCl₂, 5 glucose, and 10 HEPES (pH 7.4 with NaOH) at 22°C. The isolated tubule was transferred onto a 5 × 5-mm cover glass coated with Cell-Tak (Collaborative Research, Bedford, MA) to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon), and the tubules were superfused with HEPES-buffered NaCl solution composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.8 MgCl₂, and 10 HEPES (pH 7.4). The mTAL was cut open with a sharpened micropipette to gain access to the apical membrane. The temperature of the chamber (1,000 µl) was maintained at 37 ± 1°C by circulating warm water around the chamber.

Patch-clamp technique. An Axon 200B patch-clamp amplifier was used to record channel current. The current was low-pass filtered at 1 kHz using an eight-pole Bessel filter (902LPF; Frequency Devices, Haverhill, MA) and digitized by an Axon interface (Digitada 1200). The data 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). Opening and closing transitions were detected using 50% of the single-channel amplitude as the threshold. Channel activity was defined as NP_o, a product of channel number (N) and open probability (P_o). The NP_o was calculated from data samples of 60-s duration, which were always at the end of each experimental maneuver and in the steady state. We used the following equation to obtain NP_o

(1)

where t_i is the fractional open time spent at each of the observed current levels. Because two types of K channels are present in the apical membrane of mTAL, two types of K channels sometimes could be detected in the same patches. To avoid the complexity, we selected those patches with only a single population of K channels to calculate NP_o. If the low-conductance K channel appeared during the course of the experiments, we used a ruler to calculate NP_o manually. The mean delay time for the effect of herbimycin A or phenylarsine oxide (PAO) was ~10 min. The change in channel activity was clearly related to the application of a given chemical because the channel activity was quite stable (changes in NP_o were <10% over a 10-min period) in the absence of herbimycin A or PAO. Moreover, we have selected the results only if the effect of a given chemical was at least partially reversible.

Western blot. Protein samples extracted from the renal outer medulla were separated by electrophoresis on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline (TBS), rinsed, and washed with 1% milk in Tween-TBS. The antibodies for c-Src, PTP-1D, PTB-1B, and PTP-1C were purchased from Transduction Laboratories (Lexington, KY) and were diluted at 1:1,000. The protein concentration used for immunoblot was 50 µg. The PTPs and c-Src were detected and quantitatively analyzed by fluorescence phosphorimaging.

Chemicals and experimental solution. The pipette solution contained (in mM) 140 KCl, 1.8 mM MgCl₂, and 10 HEPES (pH 7.4). Herbimycin A and PAO were purchased from Sigma (St. Louis, MO) and dissolved in DMSO. The final concentration of DMSO was <0.1% and had no effect on channel activity. The chemicals were added directly to the bath to reach the final concentration. N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) was synthesized at J. R. Falck's laboratory, University of Texas Southwestern Medical Center at Dallas, and has been shown to specifically block cytochrome P-450 -hydroxylation of arachidonic acid (25).

Statistics. Data are shown as means ± SE. We used paired Student's t-tests to determine the significance of difference between the control and experimental periods. Statistical significance was taken as P < 0.05.

	RESULTS

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To explore the role of PTK in regulating the activity of the apical K channels in the mTAL, we investigated the effect of herbimycin A, an inhibitor of PTK, on the activity of the apical 70-pS K channel and 30-pS K channel, respectively. Figure 1A is a representative recording showing the effect of herbimycin A (1 µM) on the apical 70-pS K channel in the mTAL from rats on a KD diet. We confirmed the previous observation that channel activity was low (NP_o = 0.12 ± 0.05) in the mTAL from animals on a KD diet (9). Moreover, inhibition of PTK significantly stimulated the 70-pS K channel and increased NP_o to 0.42 ± 0.1 (n = 30) (Fig. 1, A and B). Herbimycin A also stimulated the channel activity in the mTAL from rats on a normal diet and increased NP_o modestly from 0.42 ± 0.1 to 0.56 ± 0.1 (n = 6). In contrast, the effect of herbimycin A on the 70-pS K channel is almost absent in the mTAL from rats on a HK diet. From inspection of Fig. 1B, it is apparent that inhibition of PTK slightly increased NP_o from 0.81 ± 0.16 to 0.91 ± 0.12 (n = 19). However, the difference is not significant.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   A: channel recording shows effect of 1 µM herbimycin A on activity of the apical 70-pS K channel in a cell-attached patch in the medullary thick ascending limb (mTAL) from rats on a K-deficient (KD) diet. Top trace demonstrates time course of the experiment, and 2 parts of the trace indicated by numbers are extended to show the fast-time resolution. The channel-closed level is indicated by C, and the holding potential is 0 mV. B: effect of herbimycin A on channel open probability (NPo) in the mTAL from animals on a KD diet, normal-K (NK) diet, or high-K (HK) diet. * Data are significantly different from the corresponding control value. Control bars represent channel activity obtained in absence of herbimycin A in rats on a KD, NK, or HK diet, respectively.

Because inhibition of cytochrome P-450 -oxidation has been shown to stimulate the activity of the apical 70-pS K channel in the mTAL from rats on a KD diet (9), we examined the effect of inhibiting 20-HETE production on channel activity in the presence of a PTK inhibitor. Figure 2A is a recording demonstrating that, in the presence of herbimycin A, DDMS, an agent that inhibits cytochrome P-450 -oxidation, further stimulated the activity of the 70-pS K channel. This suggests that a mechanism other than stimulating PTK is involved in mediating the inhibitory effect of 20-HETE on channel activity. Figure 2B summarizes the results of 12 experiments showing that inhibition of cytochrome P-450 monooxygenase further increased NP_o to 0.85 ± 0.1 in the presence of herbimycin A. Thus it is possible that effects of 20-HETE and PTK are mediated by at least two different signal transduction pathways.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: channel recording illustrates effects of herbimycin A (1 µM) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 5 µM) on the apical 70-pS K channels. The experiment was carried out in a cell-attached patch from the mTAL of a rat on a KD diet, and the holding potential was 0 mV. Three parts of the trace from the top recording indicated by numbers are extended to demonstrate the fast-time resolution. B: effect of herbimycin A and DDMS on NPo in the mTAL from rats on a KD diet. * Data are significantly different from control value. Control bar represents channel activity calculated in absence of herbimycin A and DDMS in rats on a KD diet.

We also examined the effect of herbimycin A on the 30-pS K channel in the mTAL from rats either on a HK diet or on a KD diet. Figure 3 summarizes the result of 20 experiments demonstrating that herbimycin A did not increase NP_o of the 30-pS K channel in the mTAL from rats on a KD diet (control, 0.21 ± 0.1; herbimycin A, 0.25 ± 0.1) or from that in rats on a HK diet (control, 0.82 ± 0.1; herbimycin A, 0.85 ± 0.1). Therefore, the result suggests that PTK does not modulate the activity of the 30-pS K channel in the mTAL.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Effect of herbimycin A (1 µM) on activity of the 30-pS K channel in the mTAL from rats on a KD diet and on a HK diet, respectively. Experiments were performed in cell-attached patches. Control represents channel activity in absence of herbimycin A in rats on a KD diet and on a HK diet, respectively.

After establishing that an increase in PTK activity may be involved in mediating the effect of low K intake on the apical 70-pS K channels in the mTAL, we examined the expression of c-Src, a nonreceptor type of PTK that is widely distributed in a variety of tissues (12). Figure 4B is a typical Western blot showing the expression of c-Src in renal outer medulla from rats on a HK diet (10 days), normal-K diet, or KD diet for 10 days. Clearly, the expression of c-Src was significantly higher in rats on a KD diet (mean increase by 190 ± 20%) than that in rats on a normal diet. Moreover, the expression of c-Src decreased significantly by 70 ± 15% (n = 4 rats) in the renal outer medulla from rats on a HK diet compared with those on a normal diet. Because the tyrosine phosphorylation is determined not only by PTK but also by PTP (12), we investigated the expression of three membrane-associated PTP isoforms, PTP-1B, PTP-1C, and PTP-1D, in the kidney from rats on a HK and on a KD diet, respectively. We failed to detect the PTP-1B and PTP-1C in the kidney (data not shown). However, PTP-1D was expressed in the renal outer medulla and cortex (Fig. 4A). Moreover, its expression was not significantly altered by K diet. This suggests that the change in the PTK expression may be an important mechanism to determine the tyrosine phosphorylation level of the 70-pS K channel in the mTAL. However, PTP can still play an important role in modifying the regulation of channel activity by PTK because the interaction of PTP and PTK determines the tyrosine phosphorylation. This notion is demonstrated by experiments in which inhibiting PTP reduced the apical 70-pS K channel activity in the mTAL from rats on a HK diet. Figure 5 is a typical recording showing the effect of PAO on the activity of the 70-pS K channel in the mTAL from rats on a HK diet. Addition of 1 µM PAO inhibited the activity of the 70-pS K channel, and NP_o dropped from 0.82 ± 0.1 to 0.2 ± 0.04 (data not shown). In contrast, PAO had no significant effect on the 30-pS K channel in the mTAL from rats on a HK diet (Fig. 6), and NP_o was almost identical (control 0.82 ± 0.1, PAO 0.81 ± 0.1). This further supports the notion that the activity of the 30-pS K channel is not regulated by PTP and PTK. The effect of PAO on the 70-pS K channel can also be observed in the mTAL from rats on a KD diet or on a normal diet (data not shown). The effect of PAO on the 70-pS K channel is produced by inhibiting PTP because application of PAO had no effect on channel activity in inside-out patches (data not shown). Moreover, herbimycin A treatment abolished the inhibitory effect of PAO (Fig. 7A). Figure 7B summarizes the results of 16 experiments showing that PAO reduced the channel activity only by 9 ± 1% from 0.95 ± 0.14 to 0.86 ± 0.12 in the presence of herbimycin in the mTAL from rats on a HK diet.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Western blots show expression of PTP-1D in the renal cortex and outer medulla from rats on a HK or KD diet (A) and expression of c-Src in the renal outer medulla in rats on a HK, NK (control), or KD diet (B). PC, positive control.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Recording showing effect of phenylarsine oxide (PAO; 1 µM) on the apical 70-pS K channel in the mTAL from a rat on a HK diet. Three parts of the trace indicated by numbers are extended to show the fast-time resolution. The pipette holding potential was 0 mV.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Recording showing effect of PAO (1 µM) on the 30-pS K channel in the mTAL from a rat on a HK diet. The experiment was carried out in a cell-attached patch, and the channel-close level is indicated by C. Top trace shows time course of the experiment, and 2 parts of the trace are demonstrated at a fast-time resolution. The holding potential was 0 mV.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   A: channel recording demonstrates the effect of PAO (1 µM) on the 70-pS K channel in the mTAL treated with herbimycin A. Top trace shows time course of the experiment, and 2 parts of the trace are demonstrated at a fast time resolution. The experiment was performed in a cell-attached patch, and holding potential was 0 mV. B: effect of PAO on the apical 70-pS K channel in the presence of herbimycin A in the mTAL from rats on a HK diet. Control bar represents channel activity in the absence of either herbimycin A or PAO in rats on a HK diet.

To explore the possibility that the 70-pS K channels are phosphorylated directly and tyrosine phosphorylation results in an inhibition of channel activity, we examined the effect of exogenous c-Src on the 70-pS K channel. Figure 8 is a channel recording showing the effect of c-Src on channel activity in an inside-out patch. Clearly, c-Src (1 nM) reversibly inhibited the 70-pS K channel and reduced NP_o by 95 ± 5% (n = 4). From inspection of Fig. 8, it is also apparent that c-Src did not affect the activity of the 30-pS K channel, which was present in the same patch.

View larger version (51K): [in this window] [in a new window]   Fig. 8.   Effect of exogenous c-Src (1 nM) on activity of the 70-pS K channel in an inside-out patch. The bath solution contains 100 nM free Ca2+ and 0.2 mM Mg ATP. Top trace shows time course of the experiment, and 4 parts of the trace indicated by numbers are extended to demonstrate the detail of channel activity. The low-conductance K channel, indicated by an arrow, coexisted in the same patch and is not affected by c-Src. The activity of c-Src is confirmed independently in measuring 32P-labeled ATP incorporation into c-Src-specific substrates.

	DISCUSSION

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The main finding of the present study is that stimulation of PTK decreased, whereas stimulation of PTP increased, the activity of the apical 70-pS K channel. The second finding is that PTP and PTK did not modulate the activity of the 30-pS K channels in the mTAL. We previously demonstrated that inhibiting PTK increased the number of low-conductance K channels in the CCD (28), a counterpart of the 30-pS K channel in the mTAL (2, 11, 27, 32). Also, we have shown that tyrosine residue 337 in the COOH terminus of ROMK1 is the key site for the effects of PTK and PTP because mutating the tyrosine residue to alanine abolished the effects of PAO and herbimycin A on ROMK1 (17). In situ hybridization study has shown that ROMK1 is located in the CCD, whereas ROMK2 and ROMK3 are expressed in the mTAL (2). Because the PTK consensus phosphorylation site (tyrosine residue 318) is also present in ROMK2, the different response to PTP and PTK must be attributed to the NH₂ terminus of ROMK2, which is 19 amino acids shorter than that of ROMK1. It is possible that the NH₂ terminus may be required for the tyrosine phosphorylation or dephosphorylation. Alternatively, the NH₂ terminus may be important for the downregulation of ROMK1 channel activity after the tyrosine phosphorylation of the channel. We have previously demonstrated that the effect of PTK on ROMK1 is the result of stimulating endocytosis, whereas the effect of PTP is mediated by exocytosis (17). Therefore, it is possible that the NH₂ terminus may be required for PTK-induced endocytosis or PTP-induced exocytosis. Relevant to the possibility that the NH₂ terminus of ROMK channels may have an important function for the channel regulation was our preceding observation that arachidonic acid blocks ROMK1 but has no effect on either ROMK2 or ROMK3 (15). Moreover, mutation of serine residue 4 [a putative protein kinase C (PKC) phosphorylation site of ROMK1] to alanine can largely abolish the effect of arachidonic acid on ROMK1. This suggests that the PKC phosphorylation site plays a key role in rendering ROMK1 the sensitivity to arachidonic acid. However, it is not known whether the PKC phosphorylation site is also essential for mediating the effect of PTK. We need further experiments to explore the role of the NH₂ terminus in mediating the effect of PTK.

In the present study, we used PAO to demonstrate the role of PTP and herbimycin A to show the effect of PTK. Three lines of evidence suggested that the effects of PAO and herbimycin A are specific for inhibiting PTP and PTK, respectively. First, the effect of herbimycin A was observed in the mTAL only in animals on a KD diet but not from those on a HK diet. Second, the effect of PAO and herbimycin A was absent in excised patches. Third, we failed to observe the stimulatory effect of herbimycin A on channel activity in the mTAL treated with 2 µM PAO (unpublished observation), whereas the effect of PAO was also abolished in the mTAL treated with herbimycin A. This suggests that the effect of the two agents is the result of altering the balance of PTP and PTK interaction.

We confirmed the previous observation that the expression of c-Src in the outer medulla of kidneys was significantly higher from rats on a KD diet than that from animals on a HK diet (28). Also, we have shown that the expression of PTP-1D was not changed in the kidney by dietary K intake. However, it is conceivable that tyrosine phosphorylation is enhanced in the mTAL from rats on a KD diet because PTK activity increased severalfold. PTK has been shown to play an important role in regulating a variety of K channels (3, 22, 31, 34). There are at least two mechanisms by which PTK modulates the channel activity. First, PTK can alter the channel activity by modifying endocytosis or recycling. It has been shown that PTK is involved in regulating the endocytosis of G protein-coupled receptors (14). Second, PTK can directly phosphorylate the ion channels and accordingly regulate the channel activity. Relevant to the first possibility is the observation that PTK decreases the number of the apical small-conductance K channels in the CCD via endocytosis (28). However, our data indicate that the effect of PTK results from a direct phosphorylation of the 70-pS K channel or its associated proteins because addition of exogenous c-Src blocks the channel. It was shown that PTK suppresses delayed rectifying K channels (22), voltage-gated K channels, Kv1.3 (3), and Ca²⁺-dependent K channels by direct tyrosine phosphorylation (20). Moreover, c-Src has been shown to regulate the N-methyl-D-aspartate channel by direct association and phosphorylation (31, 34).

There are three types of apical K channels (30-pS, 70-pS, and a Ca²⁺-activated maxi-K channel) in the apical membrane of the mTAL (1, 5, 26, 30). Moreover, the ROMK channel is the most likely pore-forming subunit of the 30-pS K channel (18). However, it still has not been established whether the ROMK channel is also a part of the 70-pS K channel. It is generally accepted that the 30-pS and 70-pS K channels are mainly responsible for K recycling. The importance of K recycling in maintaining the function of the Na-K-Cl cotransporter is best demonstrated by genetic studies in which defective gene product encoding ROMK channel results in abnormal renal salt transport (21). Hypokalemia has been shown to impair the epithelial transport in the mTAL (10, 23). However, the mechanism by which K depletion impairs the epithelial transport in the mTAL is not completely understood. Because the 70-pS K channel has an important role in K recycling, it is possible that a diminished apical K recycling is partially responsible for the impaired NaCl transport in the mTAL during hypokalemia. Moreover, PTK and PTP should be involved in regulating NaCl transport in the mTAL because they modulate the activity of the apical K channels. In this regard, it is possible that increased c-Src activity in the kidney from animals on a KD diet is partially responsible for impairing epithelial transport in the mTAL during K depletion.

We have previously demonstrated that inhibition of cytochrome P-450 monooxygenase increased channel activity (9). We have now shown that an increase in PTK activity may also be responsible for suppressing the channel activity in the mTAL. One possible mechanism by which 20-HETE inhibits the 70-pS K channel is that the 20-HETE effect may be mediated by stimulating PTK. It has been reported that the effect of 20-HETE on the Ca²⁺-activated K channel is mediated by PTK in smooth muscle cells (24). Moreover, PTK has been suggested to mediate the effect of 20-HETE on bicarbonate transport in the mTAL (7). Finally, Chen et al. (4) have demonstrated that 14,15-epoxyeicosatrienoic acid, a cytochrome P-450 epoxygenase-dependent metabolite of arachidonic acid, stimulates tyrosine phosphorylation in renal epithelial cells (4). However, the observation that inhibition of cytochrome P-450 -oxidation can further increase channel activity in the presence of herbimycin A strongly suggests that the inhibitory effect of 20-HETE on the 70-pS K channel is produced by a mechanism other than stimulating PTK. It is most likely that the effects of 20-HETE and PTK are independent, although we cannot completely exclude the possibility that the partial effect of 20-HETE may be achieved by stimulation of PTK.

We have previously demonstrated that the activity of the 70-pS K channel is inhibited by PKC (29) and activated by protein kinase A (PKA) and cGMP-dependent kinase (13). In the present study, we have shown that PTK is also an important regulator for the 70-pS K channel. Moreover, it is possible that the regulation of the 70-pS K channel by PTK may require cross-talk among PKC, protein kinase G, PKA, and PTK. Further experiments are needed to explore this possibility. Although increasing PTK activity has an important role in mediating the effect of dietary K intake on the 70-pS K channel in the mTAL, it is still not understood how a low K intake stimulates PTK activity. Because K depletion can cause intracellular acidosis that has been shown to activate c-Src (33), it is possible that intracellular acidosis is partially responsible for increasing c-Src activity during hypokalemia.

We conclude that a low K intake increases the c-Src level in the renal outer medulla. Increasing PTK activity inhibits, whereas stimulation of PTP activates, the apical 70-pS K channel in the mTAL.