Effects of 2,3-butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes.
1. We examine the actions of a chemical phosphatase, 2,3-butanedione monoxime (BDM), on endogenous and expressed Ca2+ channel currents in Xenopus oocytes. In previous studies on L-type Ca2+ channel currents in cardiomyocytes and dorsal root ganglia, the inhibitory effects of BDM were attenuated by activation of protein kinase A. 2. Ba2+ currents (IBa) through a human wild-type L-type Ca2+ channel complex (i.e. halpha1C, alpha2-deltaa and hbeta1b) are inhibited by BDM with an IC50 of 16 mM, with 10 mM producing a 36.1 +/- 2.2 % inhibition. IBa through endogenous oocyte N-type Ca2+ channels, upregulated by exogenous alpha2-deltaa and hbeta1b subunits, are inhibited to a similar degree by BDM. 3. To examine whether the action of BDM is dependent on PKA-dependent phosphorylation, a clone of halpha1C deficient in all five serine PKA consensus sites (halpha1C-SA5) was co-expressed with alpha2-deltaa and the human cardiac hbeta3 subunit, which naturally lacks PKA consensus sites. This complex exhibited a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.6 +/- 1.5 % inhibition. 4. As limited proteolysis upregulates Ca2+ channels in cardiomyocytes and renders them less sensitive to BDM, experiments were performed with a carboxyl terminus deletion mutant, halpha1C-Delta1633. IBa through this subunit showed a sensitivity to BDM that was similar to the wild-type complex, with 10 mM BDM producing 31.3 +/- 1.4 % inhibition. However, co-expression with alpha2-deltaa and hbeta3 subunits reduced potency, and is reflected by an increased IC50 of 22.7 mM. 5. The actions of BDM were examined on a rat brain rbA-1 Ca2+ channel clone, alpha1A, co-expressed with alpha2-deltab and beta1b subunit homologues from rat brain. BDM inhibited the current through this channel complex to a similar degree to that seen for cardiac wild-type channels, with 10 mM BDM causing a 33.1 +/- 3.5 % inhibition. 6. The effects of BDM were compared at two holding potentials, -80 and -30 mV, using the halpha1C-Delta1633, alpha2-deltaa and hbeta3 subunit combination. At -30 mV BDM is more potent with 10 mM BDM reducing IBa by 39.8 +/- 2.7 %, compared with 20.8 +/- 2.2 % at -80 mV. 7. The data suggest that BDM may not exert its inhibitory action by means of a chemical phosphatase effect, but by channel block. The similar potency observed between alpha1C, alpha1A and endogenous (N-type) channels may help point towards a possible site of action; differences with the carboxyl deletion mutant may help further to define a locus of interaction.
Allen
T J
TJ
Department of Pharmacology, Royal Free Hospital School of Medicine, London NW3 2PF, UK. tjallen@rfhsm.ac.uk
Mikala
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Wu
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D000818:Animals; D001464:Barium / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D015220:Calcium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D020746:Calcium Channels, L-Type; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009206:Myocardium / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D018075:RNA, Complementary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects; D017384:Sequence Deletion; D014982:Xenopus laevis
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D046911:Macromolecular Substances; D018075:RNA, Complementary; D011994:Recombinant Proteins; C004717:diacetylmonoxime; D001464:Barium; D017868:Cyclic AMP-Dependent Protein Kinases; D003931:Diacetyl