Intracellular Binding of Terfenadine Competes with Its Access to Pancreatic ß-cell ATP-Sensitive K(+) Channels and Human ether-à-go-go-Related Gene Channels

Most blockers of both hERG (human ether-à-go-go-related gene) channels and pancreatic ß-cell ATP-sensitive K(+) (K(ATP)) channels access their binding sites from the cytoplasmic side of the plasma membrane. It is unknown whether binding to intracellular components competes with binding of these substances to K(+) channels. The whole-cell configuration of the patch-clamp technique, a laser-scanning confocal microscope, and fluorescence correlation spectroscopy (FCS) were used to study hERG channels expressed in HEK (human embryonic kidney) 293 cells and K(ATP) channels from the clonal insulinoma cell line RINm5F. When applied via the pipette solution in the whole-cell configuration, terfenadine blocked both hERG and K(ATP) currents with much lower potency than after application via the bath solution, which was not due to P-glycoprotein-mediated efflux of terfenadine. Such a difference was not observed with dofetilide and tolbutamide. 37–68% of hERG/EGFP (enhanced green-fluorescent protein) fusion proteins expressed in HEK 293 cells were slowly diffusible as determined by laser-scanning microscopy in the whole-cell configuration and by FCS in intact cells. Bath application of a green-fluorescent sulphonylurea derivative (Bodipy-glibenclamide) induced a diffuse fluorescence in the cytosol of RINm5F cells under whole-cell patch-clamp conditions. These observations demonstrate the presence of intracellular binding sites for hERG and K(ATP) channel blockers not dialyzable by the patch-pipette solution. Intracellular binding of terfenadine was not influenced by a mutated hERG (Y652A) channel. In conclusion, substances with high lipophilicity are not freely diffusible inside the cell but steep concentration gradients might exist within the cell and in the sub-membrane space. GRAPHICAL ABSTRACT: [Image: see text]