The Relationship between Functional Inhibition and Binding for KCa2 Channel Blockers

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e.g. apamin) and small molecule blockers (e.g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC50s of,100 pM and,1 nM for block KCa2.2 and KCa2.3 respectively. In binding studies, however, apamin appears not to discriminate between KCa2.2 and 2.3 and is reported to have a significantly higher (,20–200-fold) affinity (,5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured 125I-apamin binding to intact HEK 293 cells expressing KCa2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e.g.125I-apamin bound to KCa2.2 with KL 91640 pM and to KCa2.3 with KL 7116126 pM, while inhibiting KCa2.2 current at IC50 10362 pM). We also examined KCa2 channel block in Ca 2+ and Mg2+ free solutions that mimic conditions reported i