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 ...