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. 1993 Jul;466:707–726.

Modification of the adenosine 5'-triphosphate-sensitive K+ channel by trypsin in guinea-pig ventricular myocytes.

T Furukawa 1, Z Fan 1, T Sawanobori 1, M Hiraoka 1
PMCID: PMC1175499  PMID: 8410713

Abstract

1. The adenosine 5'-triphosphate (ATP)-sensitive K+ channel current was recorded in guinea-pig ventricular myocytes using the patch clamp technique with inside-out patch configuration. Modification of the channel activity by intracellular application of an endoprotease trypsin was studied, and was related to a possible model of regulation of this channel. 2. Maximal ATP-sensitive K+ channel activity was observed immediately upon formation of inside-out patches in the ATP-free internal solution, thereafter activity declined both spontaneously and gradually with time; a phenomenon known as rundown. When trypsin (1 mg/ml) was applied to the intracellular side of the membrane upon formation of inside-out patches, spontaneous run-down did not occur, and this trypsin action was irreversible. Neither trypsin (1 mg/ml) applied with trypsin inhibitor (0.25 mg/ml) nor heat-denatured trypsin (1 mg/ml) could mimic this effect. When trypsin was applied to the patches after run-down, channels were reactivated at approximately 13 min. 3. Treatment with trypsin did not affect unitary current amplitude, channel gating kinetics, or sensitivity to intracellular ATP. 4. Intracellularly applied Ca2+ induced run-down of channel activity in a dose-dependent manner. In membrane patches that were treated with trypsin (1 mg/ml) for 20 min, intracellularly applied Ca2+ up to 1 mM did not induce run-down of channel activity. 5. Intracellular application of an exopeptidase, carboxypeptidase A (1 mg/ml), but not Leu-aminopeptidase (0.5 mg/ml), prevented spontaneous or Ca(2+)-induced run-down of channel activity. 6. As postulated for several other channels, such as Na+ and Ca2+ channels, there may be a possible 'chemical gate' that is responsible for run-down of this channel activity. Application of trypsin might somehow modify this 'chemical gate', resulting in prevention of spontaneous or Ca(2+)-induced run-down. This target site for trypsin may be situated on the carboxy-terminus of the channel proteins, or of associated regulatory units. Because ATP sensitivity remained intact after trypsin treatment, the trypsin-selective site for channel inhibition is not related physically to the ATP binding site.

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Selected References

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  1. Armstrong C. M., Bezanilla F., Rojas E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol. 1973 Oct;62(4):375–391. doi: 10.1085/jgp.62.4.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ashcroft F. M. Adenosine 5'-triphosphate-sensitive potassium channels. Annu Rev Neurosci. 1988;11:97–118. doi: 10.1146/annurev.ne.11.030188.000525. [DOI] [PubMed] [Google Scholar]
  3. Ashford M. L., Sturgess N. C., Trout N. J., Gardner N. J., Hales C. N. Adenosine-5'-triphosphate-sensitive ion channels in neonatal rat cultured central neurones. Pflugers Arch. 1988 Aug;412(3):297–304. doi: 10.1007/BF00582512. [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. Catterall W. A. Structure and function of voltage-sensitive ion channels. Science. 1988 Oct 7;242(4875):50–61. doi: 10.1126/science.2459775. [DOI] [PubMed] [Google Scholar]
  6. Cook D. L., Hales C. N. Intracellular ATP directly blocks K+ channels in pancreatic B-cells. Nature. 1984 Sep 20;311(5983):271–273. doi: 10.1038/311271a0. [DOI] [PubMed] [Google Scholar]
  7. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  8. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  9. Fenwick E. M., Marty A., Neher E. Sodium and calcium channels in bovine chromaffin cells. J Physiol. 1982 Oct;331:599–635. doi: 10.1113/jphysiol.1982.sp014394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Findlay I. Calcium-dependent inactivation of the ATP-sensitive K+ channel of rat ventricular myocytes. Biochim Biophys Acta. 1988 Aug 18;943(2):297–304. doi: 10.1016/0005-2736(88)90561-5. [DOI] [PubMed] [Google Scholar]
  11. Findlay I., Dunne M. J. ATP maintains ATP-inhibited K+ channels in an operational state. Pflugers Arch. 1986 Aug;407(2):238–240. doi: 10.1007/BF00580683. [DOI] [PubMed] [Google Scholar]
  12. Findlay I., Dunne M. J., Petersen O. H. ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells. J Membr Biol. 1985;88(2):165–172. doi: 10.1007/BF01868430. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Hescheler J., Trautwein W. Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes. J Physiol. 1988 Oct;404:259–274. doi: 10.1113/jphysiol.1988.sp017289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirano Y., Hiraoka M. Barium-induced automatic activity in isolated ventricular myocytes from guinea-pig hearts. J Physiol. 1988 Jan;395:455–472. doi: 10.1113/jphysiol.1988.sp016929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horie M., Irisawa H., Noma A. Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells. J Physiol. 1987 Jun;387:251–272. doi: 10.1113/jphysiol.1987.sp016572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoshi T., Zagotta W. N., Aldrich R. W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science. 1990 Oct 26;250(4980):533–538. doi: 10.1126/science.2122519. [DOI] [PubMed] [Google Scholar]
  18. Kirsch G. E., Brown A. M. Trypsin activation of atrial muscarinic K+ channels. Am J Physiol. 1989 Jul;257(1 Pt 2):H334–H338. doi: 10.1152/ajpheart.1989.257.1.H334. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lederer W. J., Nichols C. G. Nucleotide modulation of the activity of rat heart ATP-sensitive K+ channels in isolated membrane patches. J Physiol. 1989 Dec;419:193–211. doi: 10.1113/jphysiol.1989.sp017869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983 Sep 8;305(5930):147–148. doi: 10.1038/305147a0. [DOI] [PubMed] [Google Scholar]
  22. Ohno-Shosaku T., Zünkler B. J., Trube G. Dual effects of ATP on K+ currents of mouse pancreatic beta-cells. Pflugers Arch. 1987 Feb;408(2):133–138. doi: 10.1007/BF00581342. [DOI] [PubMed] [Google Scholar]
  23. Patlak J., Horn R. Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches. J Gen Physiol. 1982 Mar;79(3):333–351. doi: 10.1085/jgp.79.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Spruce A. E., Standen N. B., Stanfield P. R. Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane. Nature. 1985 Aug 22;316(6030):736–738. doi: 10.1038/316736a0. [DOI] [PubMed] [Google Scholar]
  25. Standen N. B., Quayle J. M., Davies N. W., Brayden J. E., Huang Y., Nelson M. T. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science. 1989 Jul 14;245(4914):177–180. doi: 10.1126/science.2501869. [DOI] [PubMed] [Google Scholar]
  26. Takano M., Qin D. Y., Noma A. ATP-dependent decay and recovery of K+ channels in guinea pig cardiac myocytes. Am J Physiol. 1990 Jan;258(1 Pt 2):H45–H50. doi: 10.1152/ajpheart.1990.258.1.H45. [DOI] [PubMed] [Google Scholar]
  27. Tempel B. L., Papazian D. M., Schwarz T. L., Jan Y. N., Jan L. Y. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila. Science. 1987 Aug 14;237(4816):770–775. doi: 10.1126/science.2441471. [DOI] [PubMed] [Google Scholar]
  28. Trube G., Hescheler J. Inward-rectifying channels in isolated patches of the heart cell membrane: ATP-dependence and comparison with cell-attached patches. Pflugers Arch. 1984 Jun;401(2):178–184. doi: 10.1007/BF00583879. [DOI] [PubMed] [Google Scholar]
  29. Tung R. T., Kurachi Y. On the mechanism of nucleotide diphosphate activation of the ATP-sensitive K+ channel in ventricular cell of guinea-pig. J Physiol. 1991 Jun;437:239–256. doi: 10.1113/jphysiol.1991.sp018593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zilberter Y., Burnashev N., Papin A., Portnov V., Khodorov B. Gating kinetics of ATP-sensitive single potassium channels in myocardial cells depends on electromotive force. Pflugers Arch. 1988 May;411(5):584–589. doi: 10.1007/BF00582382. [DOI] [PubMed] [Google Scholar]

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