The inhibitory action of o-vanillin could therefore be mediated via this cascade. To investigate this possibility, RBCs were pre-treated with N-ethylmaleimide
(NEM; 1 mM), a thiol-reacting reagent which activates KCC activity and abolishes its sensitivity to (de)phosphorylation [26]. Under these conditions, substantial inhibition of KCC activity by o-vanillin (5 mM) was still observed in RBCs from both HbSS and HbSC individuals (Figs. 4a & b). The IC50 for o-vanillin on KCC activity in NEM-treated RBCs from
HbSS patients was about 0.3 mM (Fig. 4c). It would therefore appear that the action of o-vanillin on KCC is not via the regulatory phosphorylation cascade but more likely directly on the transporter itself.