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

摘要

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.Ba2+ currents (IBa) through a human wild-type L-type Ca2+ channel complex (i.e. hα1C, α2-δa and hβ1b) 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 α2-δa and hβ1b subunits, are inhibited to a similar degree by BDM.To examine whether the action of BDM is dependent on PKA-dependent phosphorylation, a clone of hα1C deficient in all five serine PKA consensus sites (hα1C-SA5) was co-expressed with α2-δa 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.As limited proteolysis upregulates Ca2+ channels in cardiomyocytes and renders them less sensitive to BDM, experiments were performed with a carboxyl terminus deletion mutant, hα1C-Δ1633. 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 α2-δa and hβ3 subunits reduced potency, and is reflected by an increased IC50 of 22.7 mm.The actions of BDM were examined on a rat brain rbA-1 Ca2+ channel clone, α1A, co-expressed with α2-δb and β1b 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.The effects of BDM were compared at two holding potentials, -80 and −30 mV, using the hα1C-Δ1633, α2-δa and hβ3 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.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 α1C, α1A 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.