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

• Published in: The Journal of physiology Vol. 508; no. 1; pp. 1 - 14
• Main Authors: Allen, T. J. A., Mikala, G., Wu, X.‐P., Dolphin, A. C.
• Format: Journal Article
• Language: English
• Published: Oxford, UK The Physiological Society 01-04-1998; Blackwell Science Ltd; Blackwell Science Inc
• Subjects: Animals; Barium - pharmacology; Brain - metabolism; Calcium Channels - biosynthesis; Calcium Channels - drug effects; Calcium Channels - physiology; Calcium Channels, L-Type; Cloning, Molecular; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; Diacetyl - analogs & derivatives; Diacetyl - pharmacology; Humans; Kinetics; Macromolecular Substances; Membrane Potentials - drug effects; Membrane Potentials - physiology; Myocardium - metabolism; Oocytes - drug effects; Oocytes - physiology; Original; Rats; Recombinant Proteins - biosynthesis; Recombinant Proteins - drug effects; RNA, Complementary; Sequence Deletion; Xenopus; Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1111/j.1469-7793.1998.001br.x

We examine the actions of a chemical phosphatase, 2,3-butanedione monoxime (BDM), on endogenous and expressed Ca 2+ channel currents in Xenopus oocytes. In previous studies on L-type Ca 2+ channel currents in cardiomyocytes and dorsal root ganglia, the inhibitory effects of BDM were attenuated by activation of
protein kinase A.
Ba 2+ currents ( I Ba ) through a human wild-type L-type Ca 2+ channel complex (i.e. hα 1C , α 2 -δ a and hβ 1b ) are inhibited by BDM with an IC 50 of 16 m m , with 10 m m producing a 36.1 ± 2.2 % inhibition. I Ba through endogenous oocyte N-type Ca 2+ channels, upregulated by exogenous α 2 -δ a and hβ 1b 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 hα 1C deficient in all five serine PKA consensus sites (hα 1C-SA5 ) was co-expressed with α 2 -δ a and the human cardiac hβ 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 m m BDM producing 31.6 ± 1.5 % inhibition.
As limited proteolysis upregulates Ca 2+ 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 wild-type complex, with 10 m m BDM producing 31.3 ± 1.4 % inhibition. However, co-expression with α 2 -δ a and hβ 3 subunits reduced potency, and is reflected by an increased IC 50 of 22.7 m m .
The actions of BDM were examined on a rat brain rbA-1 Ca 2+ channel clone, α 1A , co-expressed with α 2 -δ b and β 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 m m BDM causing a 33.1 ± 3.5 % inhibition.
The effects of BDM were compared at two holding potentials, -80 and −30 mV, using the hα 1C-Δ1633 , α 2 -δ a and hβ 3 subunit combination. At −30 mV BDM is more potent with 10 m m BDM reducing I Ba 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 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.