Under these conditions, the magnitude of the CLT-sensitive K+ influx was modest, at about 6 mmol (l cells h)−1, considerably below the peak values achievable in RBCs following full activation of the channel. Using the ionophore A23187 to load RBCs with Ca2+ [28] can achieve activities of several hundred mmol (l cells h)−1. In fully oxygenated conditions, RBCs were incubated with A23187 (4 μM) and an extracellular Ca2+ of
10 μM to give a free intracellular Ca2+ of about 20 μM, given the usual Donnan ratio of about 1.4 [29]. Gardos channel activity of up to 700 mmol K+ (l cells h)− was achieved which was still largely abolished in the presence of 5 mM o-vanillin in both HbSS and HbSC RBCs (Fig. 5a). The IC50 was about 1 mM whilst inhibition at 5mMwas about 95% (Fig. 5b). Thus o-vanillin was also able to inhibit Gardos channel activity directly, and again this was irrespective of any effect on HbS polymerisation or Psickle activity