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. 1992 Aug;454:673–688. doi: 10.1113/jphysiol.1992.sp019286

Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells.

K Ono 1, H A Fozzard 1
PMCID: PMC1175627  PMID: 1335510

Abstract

1. Rundown of L-type calcium channels was studied in inside-out patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. In the cell-attached patches single-channel activity was stable for more than 15 min after the patch pipette sealed. beta-Receptor stimulation by isoprenaline caused a characteristic increase in opening probability and the appearance of prolonged openings. When the patch was excised to the inside-out configuration and exposed to a simple ionic solution, channel activity disappeared within 1-2 min and never reappeared spontaneously. 3. After rundown of L-type channel activity in the excised patch, exposure of the inside face of the patch to MgATP and the catalytic subunit of the cyclic AMP-dependent protein kinase (PKAc) resulted in recovery of Ca2+ channel activity. Under these conditions channel activity could be even greater than under control cell-attached conditions, resembling channel activity after exposure to isoprenaline. This recovery of activity persisted many minutes, usually until the patch was lost. Addition of MgATP alone caused a small transient increase in channel activity in some patches. 4. Recovery of activity by MgATP and PKAc could be prevented by prior exposure of the excised patch to protein kinase inhibitor (PKI), or it could be abruptly terminated by exposure to PKI after recovery of activity. Addition to the pipette solution of okadaic acid, a protein phosphatase inhibitor, greatly slowed rundown. These findings support the proposal that dephosphorylation is an important component of rundown, and that phosphorylation is needed for channel opening activity. 5. Single-channel conductance was not altered by patch excision, but it was reduced after exposure of the excised patch to MgATP and PKAc. Mg2+ was responsible for this effect, probably by direct channel block from the inside, and Mg2+ also caused a negative shift in the channel activation, as expected from shielding of inside fixed negative charges.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agus Z. S., Kelepouris E., Dukes I., Morad M. Cytosolic magnesium modulates calcium channel activity in mammalian ventricular cells. Am J Physiol. 1989 Feb;256(2 Pt 1):C452–C455. doi: 10.1152/ajpcell.1989.256.2.C452. [DOI] [PubMed] [Google Scholar]
  2. Armstrong D., Eckert R. Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2518–2522. doi: 10.1073/pnas.84.8.2518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belles B., Malécot C. O., Hescheler J., Trautwein W. "Run-down" of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pflugers Arch. 1988 Apr;411(4):353–360. doi: 10.1007/BF00587713. [DOI] [PubMed] [Google Scholar]
  4. Brehm P., Eckert R. Calcium entry leads to inactivation of calcium channel in Paramecium. Science. 1978 Dec 15;202(4373):1203–1206. doi: 10.1126/science.103199. [DOI] [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. Cavalié A., Ochi R., Pelzer D., Trautwein W. Elementary currents through Ca2+ channels in guinea pig myocytes. Pflugers Arch. 1983 Sep;398(4):284–297. doi: 10.1007/BF00657238. [DOI] [PubMed] [Google Scholar]
  7. Chad J. E., Eckert R. An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. J Physiol. 1986 Sep;378:31–51. doi: 10.1113/jphysiol.1986.sp016206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cukierman S., Zinkand W. C., French R. J., Krueger B. K. Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers. J Gen Physiol. 1988 Oct;92(4):431–447. doi: 10.1085/jgp.92.4.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hadley R. W., Lederer W. J. Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes. J Gen Physiol. 1991 Aug;98(2):265–285. doi: 10.1085/jgp.98.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hagiwara S., Byerly L. Calcium channel. Annu Rev Neurosci. 1981;4:69–125. doi: 10.1146/annurev.ne.04.030181.000441. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hartzell H. C., White R. E. Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes. J Gen Physiol. 1989 Oct;94(4):745–767. doi: 10.1085/jgp.94.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hescheler J., Mieskes G., Rüegg J. C., Takai A., Trautwein W. Effects of a protein phosphatase inhibitor, okadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflugers Arch. 1988 Aug;412(3):248–252. doi: 10.1007/BF00582504. [DOI] [PubMed] [Google Scholar]
  14. Imoto Y., Yatani A., Reeves J. P., Codina J., Birnbaumer L., Brown A. M. Alpha-subunit of Gs directly activates cardiac calcium channels in lipid bilayers. Am J Physiol. 1988 Oct;255(4 Pt 2):H722–H728. doi: 10.1152/ajpheart.1988.255.4.H722. [DOI] [PubMed] [Google Scholar]
  15. Irisawa H., Kokubun S. Modulation by intracellular ATP and cyclic AMP of the slow inward current in isolated single ventricular cells of the guinea-pig. J Physiol. 1983 May;338:321–337. doi: 10.1113/jphysiol.1983.sp014675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaibara M., Kameyama M. Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea-pig. J Physiol. 1988 Sep;403:621–640. doi: 10.1113/jphysiol.1988.sp017268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kameyama M., Hescheler J., Hofmann F., Trautwein W. Modulation of Ca current during the phosphorylation cycle in the guinea pig heart. Pflugers Arch. 1986 Aug;407(2):123–128. doi: 10.1007/BF00580662. [DOI] [PubMed] [Google Scholar]
  18. Kostyuk P. G., Veselovsky N. S., Fedulova S. A. Ionic currents in the somatic membrane of rat dorsal root ganglion neurons-II. Calcium currents. Neuroscience. 1981;6(12):2431–2437. doi: 10.1016/0306-4522(81)90089-0. [DOI] [PubMed] [Google Scholar]
  19. Kume H., Takai A., Tokuno H., Tomita T. Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature. 1989 Sep 14;341(6238):152–154. doi: 10.1038/341152a0. [DOI] [PubMed] [Google Scholar]
  20. Lacerda A. E., Kim H. S., Ruth P., Perez-Reyes E., Flockerzi V., Hofmann F., Birnbaumer L., Brown A. M. Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature. 1991 Aug 8;352(6335):527–530. doi: 10.1038/352527a0. [DOI] [PubMed] [Google Scholar]
  21. Lacerda A. E., Rampe D., Brown A. M. Effects of protein kinase C activators on cardiac Ca2+ channels. Nature. 1988 Sep 15;335(6187):249–251. doi: 10.1038/335249a0. [DOI] [PubMed] [Google Scholar]
  22. Lee K. S., Tsien R. W. High selectivity of calcium channels in single dialysed heart cells of the guinea-pig. J Physiol. 1984 Sep;354:253–272. doi: 10.1113/jphysiol.1984.sp015374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nilius B., Hess P., Lansman J. B., Tsien R. W. A novel type of cardiac calcium channel in ventricular cells. Nature. 1985 Aug 1;316(6027):443–446. doi: 10.1038/316443a0. [DOI] [PubMed] [Google Scholar]
  24. Nowycky M. C., Fox A. P., Tsien R. W. Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2178–2182. doi: 10.1073/pnas.82.7.2178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pelzer D., Pelzer S., McDonald T. F. Properties and regulation of calcium channels in muscle cells. Rev Physiol Biochem Pharmacol. 1990;114:107–207. doi: 10.1007/BFb0031019. [DOI] [PubMed] [Google Scholar]
  26. Poole R. C., Halestrap A. P., Price S. J., Levi A. J. The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes. Biochem J. 1989 Dec 1;264(2):409–418. doi: 10.1042/bj2640409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reuter H. Localization of beta adrenergic receptors, and effects of noradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. J Physiol. 1974 Oct;242(2):429–451. doi: 10.1113/jphysiol.1974.sp010716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reuter H., Stevens C. F., Tsien R. W., Yellen G. Properties of single calcium channels in cardiac cell culture. Nature. 1982 Jun 10;297(5866):501–504. doi: 10.1038/297501a0. [DOI] [PubMed] [Google Scholar]
  29. Romanin C., Grösswagen P., Schindler H. Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches. Pflugers Arch. 1991 Mar;418(1-2):86–92. doi: 10.1007/BF00370456. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg R. L., Hess P., Reeves J. P., Smilowitz H., Tsien R. W. Calcium channels in planar lipid bilayers: insights into mechanisms of ion permeation and gating. Science. 1986 Mar 28;231(4745):1564–1566. doi: 10.1126/science.2420007. [DOI] [PubMed] [Google Scholar]
  31. Sheets M. F., Scanley B. E., Hanck D. A., Makielski J. C., Fozzard H. A. Open sodium channel properties of single canine cardiac Purkinje cells. Biophys J. 1987 Jul;52(1):13–22. doi: 10.1016/S0006-3495(87)83183-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yamamoto D., Yeh J. Z., Narahashi T. Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels. Biophys J. 1984 Jan;45(1):337–344. doi: 10.1016/S0006-3495(84)84159-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yatani A., Codina J., Imoto Y., Reeves J. P., Birnbaumer L., Brown A. M. A G protein directly regulates mammalian cardiac calcium channels. Science. 1987 Nov 27;238(4831):1288–1292. doi: 10.1126/science.2446390. [DOI] [PubMed] [Google Scholar]
  35. Yue D. T., Herzig S., Marban E. Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Natl Acad Sci U S A. 1990 Jan;87(2):753–757. doi: 10.1073/pnas.87.2.753. [DOI] [PMC free article] [PubMed] [Google Scholar]

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