Skip to main content
Log in

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches

  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

The activity of single L-type Ca2+ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca2+ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent rundown as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca2+ channel regulation with free access to both sides of the membrane.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Armstrong DL (1989) Calcium channel regulation by calcineurin, a Ca2+-activated phosphatase, in mammalian brain. Trends Neurosci 12:117–122

    Google Scholar 

  2. Armstrong D, Eckert R (1987) Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. Proc Natl Acad Sci USA 84:2518–2522

    Google Scholar 

  3. Bechem M, Pott L (1985) Removal of Ca current inactivation in dialysed guinea-pig atrial cardioballs by Ca chelators. Pflügers Arch 404:10–20

    Google Scholar 

  4. Belles B, Hescheler J, Trautwein W, Blomgren K, Karlsson JO (1988) A possible physiological role of the Ca-dependent protease calpain and its inhibitor calpastatin on the Ca current in guinea pig myocytes. Pflügers Arch 412:554–556

    Google Scholar 

  5. Belles B, Malecot CO, Hescheler J, Trautwein W (1988) “Rundown” of the Ca current during whole-cell recording in guinea-pig heart cells. Pflügers Arch 411:353–360

    Google Scholar 

  6. Bond JS, Butler PE (1987) Intracellular proteases. Annu Rev Biochem 56:333–364

    Google Scholar 

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  8. Brum G, Osterrieder W, Trautwein W (1984) β-Adrenergic increase in the calcium conductance of cardiac myocytes studied with the patch clamp. Pflügers Arch 401:111–118

    Google Scholar 

  9. Cavalie A, Ochi R, Pelzer D, Trautwein W (1983) Elementary currents through Ca2+ channels in guinea pig myocytes. Pflügers Arch 398:284–297

    Google Scholar 

  10. Chad JE, Eckert R (1986) An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. J Physiol 378:31–51

    Google Scholar 

  11. Chen M, Stracher A (1989) In situ phosphorylation of platelet actin-binding protein by cAMP-dependent protein kinase stabilizes it against proteolysis by calpain. J Biol Chem 264:14282–14289

    Google Scholar 

  12. DeMartino EN, Croall DE (1985) Calcium-dependent proteases from liver and heart. In: Khairallah EA, Bond JS, Bird JWC (eds) Intracellular protein catabolism. Liss, New York, p 261

    Google Scholar 

  13. Dösemeci A, Dhallan RS, Cohen NM, Lederer WJ, Rogers TB (1988) Phorbol ester increases calcium current and simulates the effect of angiotensin II on cultured neonatal rat heart myocytes. Circ Res 62:347–357

    Google Scholar 

  14. Goll DE, Edmunds T, Kleese WC, Sathe SK, Shannon JD (1985) Some properties of the Ca2+-dependent proteinase. In: Khairallah EA, Bond JS, Bird JWC (eds) Intracellular protein catabolism. Liss, New York, p 151

    Google Scholar 

  15. Hamill OP, Marty A, Neher E, Sakman B, Sigworth FJ (1981) Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    Google Scholar 

  16. Hescheler J, Trautwein W (1988) Modification of L-type calcium current by intracellular applied trypsin in guinea-pig ventricular myocytes. J Physiol (Lond) 404:259–274

    Google Scholar 

  17. Hescheler J, Kameyama M, Trautwein W, Mieskes G, Sölin HD (1987) Regulation of the cardiac calcium channel by protein phosphatases. Eur J Biochem 165:261–266

    Google Scholar 

  18. Hescheler J, Mieskes G, Ruegg JC, Takai A, Trautwein W (1988) Effects of a protein phosphatase inhibitor, ocadaic acid, on membrane currents of isolated guinea-pig cardiac myocytes. Pflügers Arch 412:248–252

    Google Scholar 

  19. Hess P, Lansman JB, Tsien RW (1986) Voltage and concentration dependence of single channel current in ventricular heart cells. J Gen Physiol 88:293–319

    Google Scholar 

  20. Hosey MM, Borsotto M, Lazdunski M (1986) Phosphorylation and dephosphorylation of dihydropyridine-sensitive voltage-dependent Ca2+ channel in skeletal muscle membranes by cAMP- and Ca2+-dependent processes. Proc Natl Acad Sci USA 83:3733–3737

    Google Scholar 

  21. Imoto Y, Yatani A, Reeves JP, Codina J, Birnbaumer L, Brown AM (1988) Subunit of Gs directly activates cardiac channels in lipid bilayers. Am J Physiol 255:H722-H728

    Google Scholar 

  22. Isenberg G, Klöckner U (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a KB-medium. Pflügers Arch 395:6–18

    Google Scholar 

  23. Kameyama M, Hescheler J, Hofmann F, Trautwein W (1986) Modulation of Ca current during the phosphorylation cycle in the guinea pig heart. Pflügers Arch 407:123–128

    Google Scholar 

  24. Kameyama M, Kameyama A, Nakayama T, Kaibara M (1988) Tissue extract recovers cardiac calcium channels from ‘run-down’. Pflügers Arch 412:328–330

    Google Scholar 

  25. Krinks MH, Haiech J, Rhoads A, Klee CB (1983) Reversible and irreversible activation of cyclic nucleotide phosphodiasterase: separation of the regulatory and catalytic domains by limited proteolysis. Adv Cyclic Nucleotide Protein Phosphorylation Res 16:31–47

    Google Scholar 

  26. Lacerda AE, Rampe D, Brown AM (1988) Effects of protein kinase C activators on cardiac Ca2+ channels. Nature 335:249–251

    Google Scholar 

  27. Melloni E, Pontremoli S (1989) The calpains. Trends Neurosci 12:438–444

    Google Scholar 

  28. Melloni E, Pontremoli S, Michetti M, Sacco O, Sparatore B, Salamino F, Horecker BL (1985) Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase. Proc Natl Acad Sci USA 82:6435–6439

    Google Scholar 

  29. Pallen CJ, Valentine KA, Wang JH, Hollenberg MD (1985) Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone. Biochemistry 24:4727–4730

    Google Scholar 

  30. Romanin Ch, Größwagen P, Schindler H (1989) Stabilization of cardiac calcium channel activity in excised patches. Biophys J 55:299a

    Google Scholar 

  31. Schneider T, Hofmann F (1988) The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Eur J Biochem 174:369–375

    Google Scholar 

  32. Suzuki K (1987) Calcium activated neutral protease: domain structure and activity regulation. Trends Biochem Sci 12:103–104

    Google Scholar 

  33. Suzuki K, Imajoh S, Emori Y, Kawasaki H, Minami Y, Ohno S (1987) Calcium-activated neutral protease and its endogenous inhibitor. FEBS Lett 220:271–277

    Google Scholar 

  34. Tallant EA, Brumöey LM, Wallace RW (1988) Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease. Biochemistry 27:2205–2211

    Google Scholar 

  35. Trautwein W, Kameyama M (1986) Intracellular control of calcium and potassium currents in cardiac cells. Jpn Heart J 27 [Suppl I]:31–50

    Google Scholar 

  36. Vaghy PL, Striessnig J, Miwa K, Knaus HG, Itagaki K, McKenna E, Glossmann H, Schwartz A (1987) Identification of a novel 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide in calcium channel preparations. J Biol Chem 262:14337–14342

    Google Scholar 

  37. Walsh KB, Kass RS (1988) Regulation of a heart potassium channel by protein kinase A and C. Science 242:67–69

    Google Scholar 

  38. Wang KKW, Villalobo A, Roufogalis BD (1989) Calmodulin-binding proteins as calpain substrates. Biochem J 262:693–706

    Google Scholar 

  39. Yatani A, Codina J, Imoto Y, Reeves JP, Birnbaumer L, Brown AM (1987) Direct regulation of mammalian cardiac calcium channels by a G protein. Science 238:1288–1293

    Google Scholar 

  40. Zimmermann UJ, Schlaepfer WW (1984) Calcium-activated neutral protease (CANP) in brain and other tissues. Prog Neurobiol 23:63–78

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Romanin, C., Grösswagen, P. & Schindler, H. Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches. Pflügers Archiv 418, 86–92 (1991). https://doi.org/10.1007/BF00370456

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00370456

Key words

Navigation