, Volume 29, Issue 1, pp 32–39 | Cite as

cAMP-dependent regulation of Ca2+ channels expressed inXenopus oocytes

  • Ya. M. Shuba
  • V. G. Naidenov
  • M. Morad


Rat forebrain- and heart-derived mRNA were used to express Ca2+ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca2+ channel currents (I Ba, 40 mM Ba2+ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI Ba, suggesting that the expressed Ca2+ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca2+ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca2+ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.


Adenylate Cyclase Xenopus Oocyte Protein Kinase Inhibitor Calcium Channel Current Cardiac mRNA 
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  1. 1.
    N. Dascal, T. P. Snutch, H. Lübbert, et al., “Expression and modulation of voltage-gated calcium channels after mRNA injection inXenopus oocytes,”Science,231, 1147–1150 (1986).PubMedGoogle Scholar
  2. 2.
    S. Kaneko and Y. Nomura, “Cyclic AMP facilltates slow-inactivating Ca2+ channel current expressed byXenopus oocyte after injection of rat brain mRNA,”Neurosci. Lett.,83, 123–127 (1987).PubMedCrossRefGoogle Scholar
  3. 3.
    J. P. Leonard, J. Nargeot, T. P. Snutch et al., “Ca channels induced inXenopus oocytes by rat brain mRNA,”J. Neurosci.,7, 875–881 (1987).PubMedGoogle Scholar
  4. 4.
    P. Lory and J. Nargeot, “Cyclic AMP-dependent modulation of cardiac Ca channels expressed inXenopus laevis oocytes,”Biochem. Biophys. Res. Commun.,182, 1059–1065 (1992).PubMedCrossRefGoogle Scholar
  5. 5.
    U. Klöckner, K. Itagaki, I. Bodi, and A. Schwartz, “β-subunit expression is required for cAMP-dependent increase of cloned cardiac and vascular calcium channel currents,”Pflügers Arch,420, 413–415 (1992).PubMedCrossRefGoogle Scholar
  6. 6.
    A. Yoshida, M. Takahashi, S. Nishimura, et al., “Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel,”FEBS Lett.,309, 343–349 (1992).PubMedCrossRefGoogle Scholar
  7. 7.
    A. Sculptoreanu, E. Rotman, M. Takahashi, et al., “Voltage-dependent potentiation of the activity of cardiac L-type calcium channel α1 subunits due to phosphorylation by cAMP-dependent protein kinase,”Proc. Natl. Acad. Sci. USA,90, 10135–10139 (1993).PubMedCrossRefGoogle Scholar
  8. 8.
    D. Singer-Lahat, I. Lotan, M. Biel, et al., “Cardiac calcium channels expressed inXenopus oocytes are modulated by dephosphorylation but not cAMP-dependent phosphorylation,”Receptors Channels,2, 215–226 (1994).PubMedGoogle Scholar
  9. 9.
    E. Perez-Reyes, W. Yuan, X. Wei, and D. M. Bers, “Regulation of the cloned L-type calcium channel by cyclic-AMP-dependent protein kinase,”FEBS Lett.,342, 119–123 (1994).PubMedCrossRefGoogle Scholar
  10. 10.
    A. Yatani, M. Wakamori, T. Niidome, et al., “Stable expression and coupling of cardiac L-type Ca2+ channels with β1-adrenoreceptors,”Circ. Res.,76, 335–342 (1995).PubMedGoogle Scholar
  11. 11.
    X. Zong, J. Schreieck, G. Mehrke, et al., “On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation,”Pflügers Arch,430, 340–347 (1995).PubMedCrossRefGoogle Scholar
  12. 12.
    Y. M. Shuba, V. I. Naidenov, and M. Morad, “Glass-funnel technique for the recording of membrane currents and intracellular perfusion ofXenopus oocytes,”Pflügers Arch.,432, 562–570 (1996).PubMedCrossRefGoogle Scholar
  13. 13.
    P. Chomczynski and N. Sacchi, “Single-step method for RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction,”Analyt. Biochem.,162, 156–159 (1987).PubMedCrossRefGoogle Scholar
  14. 14.
    M. Boutjdir, P.-F. Mery, R. Hauf, et al. “High affinity forskolin inhibition of L-type Ca2+ current in cardiac cells,”Mol. Pharmacol.,38, 758–765 (1990).PubMedGoogle Scholar
  15. 15.
    T. Asai, S. Pelzer, T. F. McDonald, and D. Pelzer, “Forskolin-induced inhibition of L-type calcium channel current in isolated guinea-pig ventricular cardiomyocytes,”J. Physiol.,438, 225P (1991).Google Scholar
  16. 16.
    T. Asai, S. Pelzer, and T. F. McDonald, “Cyclic AMP-independent inhibition of cardiac calcium current by forskolin,”Mol. Pharmacol.,50, 1262–1271 (1996).PubMedGoogle Scholar
  17. 17.
    K. Kusano, R. Miledi, and J. Stinnakre, “Cholinergic and catecholaminergic receptors inXenopus occyte membrane,”J. Physiol.,328, 143–170 (1982).PubMedGoogle Scholar
  18. 18.
    C. Van Renterghem, J. Penit-Soria, and J. Stinnakre, “β-adrenergic induced K+ current inXenopus oocytes: role of cAMP, inhibition by muscarinic agents,”Proc. Roy. Soc. Lond. Ser. B,223, 389–402 (1985).CrossRefGoogle Scholar
  19. 19.
    A. A. Smith, T. Brooker, and G. Brooker, “Expression of rat mRNA coding for hormone-stimulated adenylate cyclase inXenopus oocytes,”FASEB J.,1, 380–387 (1987).PubMedGoogle Scholar
  20. 20.
    M. Kameyama, F. Hofmann, and W. Trautwein, “On the mechanism of β-adrenergic regulation of the Ca channel in guinea-pig heart,”Pflügers Arch,405, 285–293 (1985).PubMedCrossRefGoogle Scholar
  21. 21.
    E. Bourinet, F. Fournier, J. Nargeot, and P. Charnet, “EndogenousXenopus-oocyte Ca-channels are regulated by protein kinases A and C,”FEBS Lett.,299, 5–9 (1992).PubMedCrossRefGoogle Scholar
  22. 22.
    E. M. Blumenthal and L. K. Kaczmarek, “Modulation by cAMP of slowly activating potassium channel expressed inXenopus oocytes,”J. Neurosci.,12, 290–296 (1992).PubMedGoogle Scholar
  23. 23.
    E. Gershon, L. Weigl, I. Lotan, et al., “Protein kinase A reduces voltage-dependent Na+ current inXenopus oocytes,”J. Neurosci.,12, 3743–3752 (1992).PubMedGoogle Scholar
  24. 24.
    R. D. Smith and A. L. Goldin, “Protein kinase A phosphorylation enhances sodium channel currents inXenopus oocytes,”Am. J. Physiol.,263, C660-C666 (1992).PubMedGoogle Scholar
  25. 25.
    G. G. Wilson, C. A. O'Neil, A. Sivaprasadarao, et al., “Modulation by protein kinase A of a cloned rat brain potassium channel expressed inXenopus oocytes,”Pflügers Arch.,428, 186–193 (1994).PubMedCrossRefGoogle Scholar
  26. 26.
    N. Dascal, I. Lotan, E. Karni, and A. Gigi, “Calcium channel currents inXenopus oocytes injected with skeletal muscle RNA,”J. Physiol.,450, 469–490 (1992).PubMedGoogle Scholar
  27. 27.
    A. Mikami, K. Imoto, T. Tanabe, et al., “Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel,”Nature,340, 230–233 (1989).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1997

Authors and Affiliations

  • Ya. M. Shuba
    • 1
  • V. G. Naidenov
    • 1
  • M. Morad
    • 2
  1. 1.Bogomolets Institute of Physiology, International Center for Molecular PhysiologyNational Academy of Sciences of UkraineKievUkraine
  2. 2.Department of PharmacologyGeorgetown University Medical CenterUSA

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