The relevance of a point mutation at the C-terminal end of the S6 helix (F468) and the introduction of C-type inactivation in the blockage of hEag1 channels by astemizole, imipramine and dofetilide was tested. C-type inactivation decreased block by asternizole and dofetilide but not imipramine, suggesting different binding sites in the channel. F468C mutation increased IC50 for asternizole and imipramine but in contrast to HERG channels, only slightly for dofetilide. Together with measurements on recovery of blocking, our observations indicate that the mechanism of hEag1 blockage by each of these drugs is different, and suggest relevant structural differences between hEag1 and HERG channels. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved