We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 microM herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)) in the CCD from rats on a normal diet or on a KD diet. The increase in NP(o) was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP(o) per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP(o) in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K ؉ current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K ؉ current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K ؉ current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 g/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels. ROMK, a cloned inward rectifying Kϩ channel from the renal outer medulla, is a key component of the small conductance K ϩ channel identified in the thick ascending limb and cortical collecting duct (CCD) 1 (1-3). This conclusion is based on observations that the conductance, open probability, opening and closing kinetics, and pH sensitivity of ROMK are similar to that of the native small conductance K ϩ channel (1, 3, 4). Moreover, both K ϩ channels are regulated by protein kinase A and protein kinase C (5-10). A difference between the native small conductance K ϩ channel and ROMK is that ROMK is insensitive to sulfonylurea agents, whereas the native small conductance K ϩ channel is inhibited by sulfonylurea agents (11-13). Three isoforms of ROMK, ROMK1, -2, and -3, have been found in the rat kidney (14). Based on in situ hybridization, ROMK1 is located in the apical membrane of principal cells in the CCD, whereas ROMK2 and -3 are expressed at the thick ascending limb (14). The principal cell in the CCD is responsible for Na ϩ reabsorption and K ϩ secretion, which takes place by K ϩ entering the cell across the basolateral membrane via Na,K-ATPase followed by diffusion into the lumen across the apical membrane through ROMK1-like channels (15).We have previously demonstrated that inhibition of PTP reduced the activity of the small conductance K ϩ channel in the apical membrane of the CCD of rat kidney (16). Moreover, we have reported that blocking ...

We have previously shown that inhibiting proteintyrosine kinase increased whereas inhibiting proteintyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylationinduced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamindependent internalization of the channel. ROMK11 is located in the apical membrane of the cortical collecting duct (CCD) and is generally believed to be a key component of the native small conductance K ϩ (SK) channel (2-6). The SK channels are the major contributors to the apical K ϩ conductance and are responsible for K ϩ secretion (4, 7). One important factor for regulating K ϩ secretion is the dietary K ϩ intake; a high K ϩ intake increases whereas a low K ϩ intake decreases K ϩ secretion (7). The low K ϩ intake-induced decrease in K ϩ secretion is at least partially achieved by reducing the number of SK channels in the apical membrane of the CCD (8).Our preceding experiments strongly indicated that the low K ϩ intake-induced decrease in SK channel number was mediated by protein-tyrosine kinase (PTK). This conclusion is supported by the observation that inhibition of PTK increased the number of the SK channels in the apical membrane of the CCD from rats on a K ϩ -deficient diet (8). In contrast, inhibition of PTP decreased the number of SK channels in the CCD from rats on a high K ϩ diet (9). Because the effect of inhibiting PTP on channel activity was blocked by 20% sucrose, we speculated that inhibiting PTP increases the endocytosis of the SK channels whereas inhibiting PTK augments the exocytosis of the SK channels into the cell membrane. This notion is supported by observations that inhibiting PTP with PAO reduced whereas inhibiting PTK with herbimycin A increased the membrane location of ROMK1 in oocytes injected with GF...

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K ؉ (SK) channels in the cortical collecting duct (CCD) from rats on a K ؉ -deficient diet (1). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K ؉ diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 M phenylarsine oxide (PAO) or 1 mM benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP o reversibly from control value 3.20 to 0.40. Addition of 1 M herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K ؉ diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.

Kcnj10 encodes the inwardly rectifying K(+) channel 4.1 (Kir4.1) and is expressed in the basolateral membrane of late thick ascending limb, distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). In the present study, we perform experiments in postneonatal day 9 Kcnj10(-/-) or wild-type mice to examine the role of Kir.4.1 in contributing to the basolateral K(+) conductance in the CNT and CCD, and to investigate whether the disruption of Kir4.1 upregulates the expression of the epithelial Na(+) channel (ENaC). Immunostaining shows that Kir4.1 is expressed in the basolateral membrane of CNT and CCD. Patch-clamp studies detect three types of K(+) channels (23, 40, and 60 pS) in the basolateral membrane of late CNT and initial CCD in wild-type (WT) mice. However, only 23- and 60-pS K(+) channels but not the 40-pS K(+) channel were detected in Kcnj10(-/-) mice, suggesting that Kir.4.1 is a key component of the 40-pS K(+) channel in the CNT/CCD. Moreover, the depletion of Kir.4.1 did not increase the probability of finding the 23- and 60-pS K(+) channel in the CNT/CCD. We next used the perforated whole cell recording to measure the K(+) reversal voltage in the CNT/CCD as an index of cell membrane potential. Under control conditions, the K(+) reversal potential was -69 mV in WT mice and -61 mV in Kcnj10(-/-) mice, suggesting that Kir4.1 partially participates in generating membrane potential in the CNT/CCD. Western blotting and immunostaining showed that the expression of ENaCβ and ENaCγ subunits from a renal medulla section of Kcnj10(-/-) mice was significantly increased compared with that of WT mice. Also, the disruption of Kir4.1 increased aquaporin 2 expression. We conclude that Kir4.1 is expressed in the CNT and CCD and partially participates in generating the cell membrane potential. Also, increased ENaC expression in medullary CD of Kcnj10(-/-) mice is a compensatory action in response to the impaired Na(+) transport in the DCT.