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

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Abstract
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.

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