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Effects of 2,3‐butanedione monoxime (BDM) on calcium channels expressed in Xenopus oocytes
Author(s) -
Allen T. J. A.,
Mikala G.,
Wu X.P.,
Dolphin A. C.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.001br.x
Subject(s) - xenopus , protein subunit , protein kinase a , phosphatase , biology , oocyte , phosphorylation , potassium channel , microbiology and biotechnology , biochemistry , chemistry , biophysics , embryo , gene
1 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. 2 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 mM, with 10 mM 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. 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‐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 mM BDM producing 31.6 ± 1.5 % inhibition. 4 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 mM 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 mM. 5 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 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 , α 2 ‐δ a and hβ 3 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 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.