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

T. J. A. Allen, G. Mikala *, X.-P. Wu and A. C. Dolphin

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_1C, ₂-_a and h_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 ₂-_a and h_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_1C deficient in all five serine PKA consensus sites (h_1C-SA5) was co-expressed with ₂-_a and the human cardiac h₃ 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_1C-1633. I_Ba 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 ₂-_a and h₃ 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, _1A, co-expressed with ₂-_b and _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_1C-1633, ₂-_a and h₃ subunit combination. At -30 mV 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 _1C, _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.

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