Figure 4.
PD-induced enhancement of hERG1 current indicates cooperative subunit interactions. (A) Representative current traces recorded at 0 mV for concatenated tetramers containing zero to three WT subunits together with one to four L646E subunits, before and after 10 µM PD. (B) Effect of 10 µM PD on voltage dependence of channel activation. Itail was measured at −70 mV after pulses to the indicated Vt and normalized to Itail-max under control conditions for each channel type (n = 7–15). The symbol legend between B and E refers to data plotted in B and D–F. Control Itail-Vt relationship (black pentagons) overlaps data for LE4 channels and represents the mean of all channel types. The V0.5act and z values obtained from fitting Itail-Vt relationships to a Boltzmann function (smooth curves) before and after 10 µM PD are presented in Table 1. (C) Pulse protocol (top) used to measure fully activated Itail-Vret relationship and representative currents for WT4 channels under control conditions and after 10 µM PD as indicated. (D) Effect of 10 µM PD on Itail-Vret relationships (n = 4–9) normalized to Itail at −120 mV under control conditions for LEn/WT4−n tetramers (n = 0–4). (E) [PD]-response relationships for fold increase in Itail-max at −70 mV for different tetramers according to symbol legend shown between B and E. Data were fitted with a logistic equation to determine EC50 values and Hill coefficient, nH (see Fig. 5). (F) Plot of ΔG versus number of WT subunits contained in a concatenated tetramer. ΔG was equal to −RTlnKeq, where Keq was defined as Po/(1 − Po) at maximal effect of PD and assuming maximum Po in the absence of drug to be 0.5. Linear regression analysis was used to fit calculated data (colored symbols; solid line: y = −0.39x + 0.058; R2 = 0.96) and the relationships predicted for independent subunit transitions (▾) and cooperative subunit interactions, models 1 (▴) and 2 (•). Data are expressed as mean ± SEM (n = number of oocytes).