Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K⁺ channels in the TAL

Abstract

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP(o) of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP(o) of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion

“…Because all ROMK iso- forms (1-3) have a C-terminal PTK phosphorylation motif, the reason for the isoform specificity of regulation by PTK likely involves the unique ROMK1 NH 2 terminus. Consistent with this view, the 30 pS K ϩ channel formed by ROMK2 and ROMK3 in TAL cells is not regulated by this kinase (33) and absence of high K stimulated 30 pS K ϩ channel activity in Romk1 Ϫ/Ϫ mice ( Table 6). The unique structural difference between ROMK1 and ROMK2 is that ROMK1 extended N-terminal with specific binding sites of hormones and protein kinase.…”

Acute application of TNF stimulates apical 70-pS K+channels in the thick ascending limb of rat kidney

“…Several factors that have been shown to stimulate the 70-pS K ϩ channel include protein kinase A (PKA), the NO-cGMP-dependent pathway, and PTP (12,19,29). Therefore, we examined the effect of TNF on the 70-pS K ϩ channel in the presence of PKA inhibitors (Fig.…”

“…We previously observed that the 70-pS K ϩ channel is regulated by PTK and PTP (12). If the effect of TNF was the result of stimulation of PTP, which leads to enhancement of tyrosine dephosphorylation, inhibition of PTK should mimic the effect of TNF such that the effect of TNF on the 70-pS K ϩ channel activity should be absent in the presence of a PTK inhibitor such as herbimycin A.…”

“…PTK and PTP have been shown to play important roles in the regulation of the 70-pS K ϩ channel: stimulation of PTK decreases, whereas an increase in PTP activity augments activity of the 70-pS K ϩ channel (12). The effect of PTK on the 70-pS K ϩ channel is possibly the result of the stimulation of tyrosine phosphorylation of the 70-pS K ϩ channel or its closely associated proteins because addition of exogenous c-Src PTK could inhibit the 70-pS K ϩ channel in insideout patches (12).…”

“…In addition, it is possible that ROMK is an important component of both 70-and 30-pS K ϩ channels because neither K ϩ channel can be found in ROMK knockout mice (18). We showed that apical K channels are regulated by a variety of signaling pathways, including nitric oxide (NO), protein kinase C, and protein tyrosine phospatase (PTP) (12,19,32). TNF has been shown to activate these same pathways (1,5,16,20,35).…”