Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na+/Ca2+ exchange current in guinea-pig cardiac ventricular myocytes

1 The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', o n Na+/Ca2+ exchange current (I-NCX) was investigated using the whole-cell v oltage-clamp technique in single guinea-pig cardiac ventricular myocytes an d in CCL39 fibroblast cells expressing canine NCX1. 2 I-NCX was identified as a current sensitive to KB-R7943, a relatively sel ective NCX inhibitor, at 140 mM Na+ and 2 mM Ca2+ in the external solution and 20 mM Na+ and 433 nM free Ca2+ in the pipette solution. 3 In guinea-pig ventricular cells, BDM inhibited I-NCX in a concentration-d ependent manner. The IC50 value was 2.4 mM with a Hill coefficients of 1. T he average time for 50% inhibition by 10 mpd BDM was 124 +/- 31 s (n = 5). 4 The effect of BDM was not affected by 1 muM okadaic acid in the pipette s olution, indicating that the inhibition was not via activation of okadaic a cid-sensitive protein phosphatases. 5 Intracellular trypsin treatment via the pipette solution significantly su ppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6 PAM (pralidoxime), another oxime compound, also inhibited I-NCX in a mann er similar to BDM. 7 Isoprenaline at 50 muM and phorbol 12-myristate 13-acetate (PMA) at 8 muM did not reverse the inhibition of I-NCX by BDM. 8 BDM inhibited I-NCX in CCL39 cells expressing NCX1 and in its mutant in w hich its three major phosphorylatable serine residues were replaced with al anines. 9 We conclude that BDM inhibits I-NCX but the mechanism of inhibition is no t by dephosphorylation of the Na+/Ca2+ exchanger as a 'chemical phosphatase '.