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 (I-Ba) through a human wild-type L-type Ca2+ channel complex (i.e. h alpha(1C), alpha(2)-delta(a) and h beta(1b)) are inhibited by BDM with an IC50 of 16 mM, with 10 mM producing a 36.1 + 2.2% inhibition. I-Ba through endogenous oocyte N-type Ca2+ channels, upregulated by exogenous alpha(2)-delta(a) and h beta(1b) 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 h alpha(1C) deficient in all five serine PKA consensus sites (h alpha(1C-SA5)) was co-expressed with alpha(2)-delta(a) and the human cardiac h beta(3) 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, h alpha(1C-Delta 1633.)I(Ba) through this subunit showed a sensitivity to BDM that was similar to the wildtype complex, with 10 mar BDM producing 31.3 +/- 1.4% inhibition. Hovvever, co-expression with alpha(2)-delta(a) and h beta(3) 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, alpha(1A), co-expressed with alpha(2)-delta(b) and beta(1b) 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 h alpha(1C.Delta 1633), alpha(2)-delta(a) and h beta(3) subunit combination. At -30 mV BDM is more potent with 10 mna BDM reducing I-Ba 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 alpha(1C), alpha(1A), 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.