The relationship between functional inhibition and binding for K(Ca)2 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 (125)I-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.(125)I-apamin bound to KCa2.2 with K L 91±40 pM and to KCa2.3 with K L 711±126 pM, while inhibiting KCa2.2 current at IC50 103±2 pM). We also examined KCa2 channel block in Ca(2+) and Mg(2+) free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of KCa2.2 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.

Meta-substituted aryl(thio)ethers as potent partial agonists (or antagonists) for the histamine H3 receptor lacking a nitrogen atom in the side chain.

4-(3-Aryloxypropyl)-1H-imidazoles, which possess a meta-positioned substituent in the aryl ring, have been synthesized and tested for activity at histamine H(3) receptors. The compounds having a CN, Me, or Br substituent were found to be antagonists, whereas CF(3), Et, i-Pr, t-Bu, COCH(3), or NO(2) substituents remarkably afforded partial agonists when tested in vitro on rat cerebral cortex synaptosomes for inhibition of [(3)H]histamine release. The compounds were also active in vivo, and furthermore, the CF(3)-substituted compound trifluproxim (UCL 1470, 7) acted as a potent full agonist in vivo, having ED(50) = 0.6 +/- 0.3 mg/kg per os in mice for inhibition of brain N(tau)-methylhistamine formation. Related structures have also been investigated; homologues 4-[4-(3-(trifluoromethyl)phenoxy)butyl]-1H-imidazole and 4-[2-(3-(trifluoromethyl)phenylthio)ethyl]-1H-imidazole are shown to be partial agonists, whereas the O isostere 4-[2-(3-(trifluoromethyl)phenoxy)ethyl]-1H-imidazole is an antagonist as is the S homologue 4-[3-(3-(trifluoromethyl)phenylthio)propyl]-1H-imidazole and its CH(2) isostere 4-[4-(3-(trifluoromethyl)phenyl)butyl]-1H-imidazole.