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 K1 current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K1 current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K1 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 mg/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.

Wei, Yuan, Peter Bloom, Daohong Lin, Ruimin Gu, and Wen Hui Wang. Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase. Am J Physiol Renal Physiol 281: F206–F212, 2001.-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 patchclamp 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 mM herbimycin A increased NPo, a product of channel number (N) and open probability (Po) in the CCD from rats on a normal diet or on a KD diet. The increase in NPo 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 NPo 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 NPo 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.