The Relationship Between Functional Inhibition And Binding For K(Ca)2 Channel Blockers

Small conductance calcium-activated potassium channels (K(Ca)2.1,2.2,2.3) 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 IC(50)s of similar to 100 pM and similar to 1 nM for block K(Ca)2.2 and K(Ca)2.3 respectively. In binding studies, however, apamin appears not to discriminate between K(Ca)2.2 and 2.3 and is reported to have a significantly higher (similar to 20-200-fold) affinity (similar to 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 I-125-apamin binding to intact HEK 293 cells expressing K(Ca)2 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. I-125-apamin bound to K(Ca)2.2 with K-L 91 +/- 40 pM and to K(Ca)2.3 with K-L 711 +/- 126 pM, while inhibiting K(Ca)2.2 current at IC50 103 +/- 2 pM). We also examined K(Ca)2 channel block in Ca2+ and Mg2+ free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of K(Ca)2.2 was reduced to 20 +/- 3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.