Competitive antagonism of the mouse 5-hydroxytryptamine₃ receptor by bisindolylmaleimide I, a 'selective' protein kinase C inhibitor

We examined the effects of several protein kinase C (PKC) inhibitors on the murine 5-hydroxytryptamine₃ (5-HT₃) receptor to determine whether they acted directly on the receptor. The 5-HT-evoked currents in Xenopus laevis oocytes expressing the recombinant 5-HT₃ receptor were measured with the two-electrode voltage-clamp technique. The PKC inhibitors bisindolylmaleimide I (BIM, GF109203x) and staurosporine, but not calphostin C or chelerythrine, decreased the 5-HT₃ receptor-mediated currents when coapplied with 5-HT. BIM blocked 0.5 μM 5-HT-elicited currents with an IC₅₀ value of 7 nM, whereas in the presence of 5 μM staurosporine, 42% inhibition of 0.5 μM 5-HT- mediated currents was observed. Increasing concentrations of BIM resulted in a rightward shift of the 5-HT concentration-response curve, without altering efficacy. A Schild plot was generated, which had a slope of -1.01, suggesting competitive antagonism. The K(i) value of BIM was determined to be 29 nM. To confirm competitive antagonism, a competitive binding assay was performed on Sf21 insect cells infected with the mouse 5-HT₃ receptor cDNA in a baculovirus expression vector. BIM completely displaced binding of the selective 5-HT₃ receptor antagonist [³H]GR65630. BIM bound to the 5-HT₃ receptor with a K(i) value of 61 nM, which was slightly less potent than that of the selective 5-HT₃ receptor antagonist MDL72222 (27 nM). The PKC inhibitor BIM is a potent competitive antagonist at the 5-HT₃ receptor.