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. 1994 Aug 16;91(17):8077–8081. doi: 10.1073/pnas.91.17.8077

Regulation of ROMK1 K+ channel activity involves phosphorylation processes.

C M McNicholas 1, W Wang 1, K Ho 1, S C Hebert 1, G Giebisch 1
PMCID: PMC44548  PMID: 8058760

Abstract

An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.

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Selected References

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  1. Ashcroft F. M., Ashcroft S. J., Harrison D. E. Effects of 2-ketoisocaproate on insulin release and single potassium channel activity in dispersed rat pancreatic beta-cells. J Physiol. 1987 Apr;385:517–529. doi: 10.1113/jphysiol.1987.sp016505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashcroft F. M., Kakei M. ATP-sensitive K+ channels in rat pancreatic beta-cells: modulation by ATP and Mg2+ ions. J Physiol. 1989 Sep;416:349–367. doi: 10.1113/jphysiol.1989.sp017765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belles B., Hescheler J., Trautwein W., Blomgren K., Karlsson J. O. A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes. Pflugers Arch. 1988 Oct;412(5):554–556. doi: 10.1007/BF00582548. [DOI] [PubMed] [Google Scholar]
  4. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carl A., Kenyon J. L., Uemura D., Fusetani N., Sanders K. M. Regulation of Ca(2+)-activated K+ channels by protein kinase A and phosphatase inhibitors. Am J Physiol. 1991 Aug;261(2 Pt 1):C387–C392. doi: 10.1152/ajpcell.1991.261.2.C387. [DOI] [PubMed] [Google Scholar]
  6. Findlay I. ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations. Pflugers Arch. 1987 Oct;410(3):313–320. doi: 10.1007/BF00580282. [DOI] [PubMed] [Google Scholar]
  7. Findlay I. The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in a rat insulin-secreting cell line. J Physiol. 1987 Oct;391:611–629. doi: 10.1113/jphysiol.1987.sp016759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Glantz S. B., Li Y., Rubin C. S. Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton. J Biol Chem. 1993 Jun 15;268(17):12796–12804. [PubMed] [Google Scholar]
  9. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  10. Ho K., Nichols C. G., Lederer W. J., Lytton J., Vassilev P. M., Kanazirska M. V., Hebert S. C. Cloning and expression of an inwardly rectifying ATP-regulated potassium channel. Nature. 1993 Mar 4;362(6415):31–38. doi: 10.1038/362031a0. [DOI] [PubMed] [Google Scholar]
  11. Hwang T. C., Horie M., Gadsby D. C. Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart. J Gen Physiol. 1993 May;101(5):629–650. doi: 10.1085/jgp.101.5.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ishihara H., Martin B. L., Brautigan D. L., Karaki H., Ozaki H., Kato Y., Fusetani N., Watabe S., Hashimoto K., Uemura D. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun. 1989 Mar 31;159(3):871–877. doi: 10.1016/0006-291x(89)92189-x. [DOI] [PubMed] [Google Scholar]
  13. Kozlowski R. Z., Ashford M. L. ATP-sensitive K(+)-channel run-down is Mg2+ dependent. Proc R Soc Lond B Biol Sci. 1990 Jun 22;240(1298):397–410. doi: 10.1098/rspb.1990.0044. [DOI] [PubMed] [Google Scholar]
  14. Misler S., Giebisch G. ATP-sensitive potassium channels in physiology, pathophysiology, and pharmacology. Curr Opin Nephrol Hypertens. 1992 Oct;1(1):21–33. doi: 10.1097/00041552-199210000-00005. [DOI] [PubMed] [Google Scholar]
  15. Nakashima H., Kakei M., Tanaka H. Activation of the ATP-sensitive K+ channel by decavanadate in guinea-pig ventricular myocytes. Eur J Pharmacol. 1993 Mar 23;233(2-3):219–226. doi: 10.1016/0014-2999(93)90053-k. [DOI] [PubMed] [Google Scholar]
  16. Ribalet B., Ciani S., Eddlestone G. T. ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines. J Gen Physiol. 1989 Oct;94(4):693–717. doi: 10.1085/jgp.94.4.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shenolikar S., Nairn A. C. Protein phosphatases: recent progress. Adv Second Messenger Phosphoprotein Res. 1991;23:1–121. [PubMed] [Google Scholar]
  18. Takano M., Noma A. The ATP-sensitive K+ channel. Prog Neurobiol. 1993 Jul;41(1):21–30. doi: 10.1016/0301-0082(93)90039-u. [DOI] [PubMed] [Google Scholar]
  19. Wang W. H., Giebisch G. Dual modulation of renal ATP-sensitive K+ channel by protein kinases A and C. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9722–9725. doi: 10.1073/pnas.88.21.9722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang W., Giebisch G. Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct. J Gen Physiol. 1991 Jul;98(1):35–61. doi: 10.1085/jgp.98.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]

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