were performed on HEK-293 cells expressing wild-type or mutant hERG channels. Whole-cell patch clamp measurements of I hERG were made at 37 C and data are presented as mean 5 SEM, nR5. Commercially synthesized Cavalli-2 suppressed hERG tail currents (I hERG) with a half-maximal inhibitory concentration (IC 50) of 35.6 5 1.0 nM. Aromatic residues on the S6 helix are known to be structural determinants for high affinity hERG channel block. Mutation of Y652 residues to alanine markedly reduced the affinity of Cavalli-2 for hERG (100 nM Cavalli-2 caused 61.5 5 5.0% block of WT I hERG but only 23.0 5 4.1% of Y652A I hERG). The pore helical residues T623 and S624 have been reported to make important interactions with polar aromatic para-substituents of drugs. Despite the absence of polar para-substituents in the minimal hERG blocker, T623A I hERG (which required measuring inward tail currents in high [K þ ] at À120 mV), was blocked 47.5 5 4.2% by 100 nM Cavalli-2 compared to 73.8 5 7.1% of inward WT I hERG and S624A I hERG was blocked by 29.1 5 3.8% (compared to 61.5 5 5.0% WT I hERG). Combined with computational modelling of interactions of Cavalli-2 with the hERG pore, these results support binding modes in which S624 stabilises the protonated amino group of Cavalli-2 near the internal K binding site and T623 may promote favourable pore geometries for drug binding, possibly by stabilising optimal conformations of Y652.
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