Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase

Abstract

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 microg/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.

FIG. 1. A Western blot shows the expression of cSrc and PTP-1D in oocytes injected with cSrc + ROMK1 (lanes 1 and 3) and in oocytes injected with ROMK1 alone (lanes 2 and 4)

The protein concentration in lanes 1 and 2 was 5 μg, whereas in lanes 3 and 4 it was 10 μg. PC, positive control.

FIG. 2

A, a recording showing the effect of 1 μM herbimycin A on the K⁺ current in oocytes expressing ROMK1 and cSrc. The current was measured with a two-electrode voltage clamp. At the end of the experiment, Ba²⁺ (arrow) was added to determine the Ba²⁺-sensitive K⁺ current. I, current. B, a summary showing the time course of the effect of herbimycin A on the normalized channel activity in oocytes expressing ROMK1 alone or ROMK1 + cSrc. The asterisks indicate that the differences are significant in comparison with the corresponding values obtained in oocytes injected with ROMK1 alone.

FIG. 3

A, a recording showing the effect of 1 μM PAO on the K⁺ current in oocytes expressing ROMK1 and cSrc. After washout of PAO, the pen speed of the paper recorder was set to zero and resumed to 1 cm/2 mm 10 min later. This stop-go causes a jump of the trace shown in the figure because of an increase in K⁺ current. I, current. B, a summary showing the time course of the effect of PAO on the normalized channel activity in oocytes expressing ROMK1 alone or ROMK1 + cSrc. The asterisks indicate that the differences are significant in comparison with the corresponding values obtained in oocytes injected with ROMK1 alone.

FIG. 4

A, the effect of 1 μM herbimycin A on K⁺ current in oocytes expressing ROMK1 (R1WT) + cSrc (filled circles) and the ROMK1 mutant (R1Y337A) + cSrc (open circles), respectively. Asterisks indicate that the difference between two groups is significant. B, the effect of 1 μM PAO on K⁺ current in oocytes expressing R1WT + cSrc (filled circles) and R1Y337A + cSrc (open circles), respectively.

FIG. 5. A channel recording demonstrating the effect of exogenous cSrc (1 nM) on the activity of ROMK1 channels expressed in Xenopus oocytes

The experiment was performed in an inside-out patch, and the holding potential was 0 mV. The channel closed state is indicated by C and a dotted line. Three parts of the trace indicated by numbers are extended to show the fast time resolution.

FIG. 6

A, a recording illustrating the effect of 2 μM PAO on the Ba²⁺-sensitive K⁺ current in oocytes expressing ROMK1 + cSrc in the presence or absence of 20% sucrose. The zero current is indicated by a dotted line. I, current. B, the time course of the effect of PAO on K⁺ current in the presence of 20% sucrose (open circles) and in the absence of sucrose (filled circles). Asterisks indicate that the difference between two groups is significant.

FIG. 7. A recording showing the effect of 1 μM PAO + 10 μM colchicine (A) and of 1 μM herbimycin A + colchicine (B) on K⁺ current.

The zero current is indicated by a dotted line.I, current.

FIG. 8

A, a current-voltage curve showing the Ba²⁺-sensitive K⁺ current measured with a two-electrode voltage clamp in oocytes injected with GFP-ROMK1. The bath solution contained 150 mM KCl. I, current. B, a single channel recording in a cell-attached patch from oocytes injected with GFP-ROMK1. The bath solution contained 145 mM NaCl and 5 mM KCl.

FIG. 9. The effect of PAO (1 μM) on GFP-ROMK1 distribution in the cell membrane in oocytes expressing GFP-ROMK1 alone (A) or cSrc + GFP-ROMK1 (B)

The top picture was taken immediately after adding PAO (control), and the bottom image was collected 30 min after applying PAO. The bar length represents 60 μm. C, the effect of PAO on the fluorescence intensity of GFP-ROMK1 in the oocyte membrane (arbitrary unit).

FIG. 10. The effect of herbimycin A (1 μM) on GFP-ROMK1 distribution in the cell membrane in oocytes expressing GFPROMK1 alone (A) or cSrc + GFP-ROMK1 (B)

The bar length represents 60 μm. The top image was taken immediately after adding herbimycin A, and the bottom picture was collected 30 min after applying herbimycin A. C, the effect of herbimycin A on the fluorescence intensity of GFP-ROMK1 in the oocyte membrane (arbitrary units).

FIG. 11. The effect of 1 μM PAO or 1 μM herbimycin A on K⁺ current in oocytes expressing ROMK1 + cSrc

The oocytes were pretreated with herbimycin A or PAO for 20 min. The current was measured with a two-electrode voltage clamp.I, current.

FIG. 12

A cell model illustrating the mechanisms by which PTK (A) and PTP (B) regulate ROMK1 channels.