Effects of 2,3-butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes

Abstract

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. hα(1c),α2-δ(a) and hβ3) 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 α2-δ(a) and hβ(1β) 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 hα(1c) deficient in all five serine PKA consensus sites (hα(1c-δA5a)was co-expressed with α2-δ(a) and the human cardiac hβ3 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, hα(1c-Δ1633) I(Ba) through this subunit showed a sensitivity to BDM that was similar to the wildtype complex, with 10 mM. BDM producing 31.3 ± 1.4% inhibition. However, co-expression with α2-δ(a) and hβ3, 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, a(1A), co-expressed with α2-δ(a) and hβ3 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 hα(1c-Δ1633), α2-δ(a) and hβ3, subunit combination. At -30 mM BDM is more potent with 10 mM 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 α(1c), α(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.