Abstract 1099: Dissociation of hERG Channel Block from Pharmacological Correction in a Trafficking-deficient LQT2 Mutation.
Long QT syndrome type 2 (LQT2) is caused by mutations in the human Ether-a-go-go Related Gene (hERG). Many LQT2 mutations disrupt hERG channel protein trafficking, preventing the immature protein from maturing and reaching the cell surface. For many mutations, trafficking is restored by drugs (pharmacological correction) that cause high affinity block of hERG current (IhERG). These drugs are postulated to interact with aromatic residues Y652 and F656 in the pore region. While pharmacological correction of trafficking deficient channels has therapeutic potential for treating some patients with LQT2, it is important to demonstrate whether drug block of the channel can be dissociated from pharmacological correction. We tested the hypothesis that mutations in Y652 and F656 would similarly alter drug block and pharmacological correction of a trafficking deficient LQT2 channel. We first measured IhERG from HEK293 cells transiently transfected with WT-, Y652A-, or F656A-hERG and perfused increasing concentrations of E4031 (0.01–10μM). Peak tail IhERG were fit with a Hill equation and show IC50 mean values of 0.022μM for WT, 0.549μM for Y652A, and 0.817μM (n=3–5 cells). We next studied the trafficking-deficient LQT2 mutation N470D for pharmacological correction with 0.01–10μM E4031. Cells were transiently transfected with N470D-hERG or double mutations N470D/Y652A- and N470D/F656A-hERG and incubated for 24hrs in the absence or presence of E4031. Subsequently, cells were lysed for Western Blot or E4031 was washed out for 1hr before recording IhERG. For Western Blot analysis the density of the 155kDa mature hERG protein band was normalized to maximal corrected density. Densitometry results were fit to a Hill equation yielding 50% maximal correction density of 1.12μM for N470D, 1.94μM for N470D/Y652A, and 1.34μM for N470D/F656A (n=3 blots each). Pharmacological correction of IhERG was similar for cells expressing N470D, N470D/Y652A, N470D/F656A (n=7– 8 cells). Our results indicate that for the N470D LQT2 mutation pharmacological correction of hERG trafficking can be dissociated from drug block of IhERG. These findings support the idea that distinct binding sites mediate drug block and pharmacological correction.