(hERG1a), 0.3560.01 (hERG1a/b, P,0.05 compared to hERG1a) and 0.3760.01 (hERG1b). To verify the relationship between ‘peak potential’ and relative abundance of hERG1b we repeated the experiment in X. laevis oocytes. In the oocyte expression system, cRNA is injected directly into the oocytes and hence, the relative amounts of cRNA can be controlled very precisely. In figure 3A currents recorded from oocytes injected with different ratios of hERG1a and hERG1b (indicated as % of hERG1b abundance) are shown. The voltage protocol and normalization of data are identical to what is described above for figure 2A. The data show that as the relative abundance of hERG1b was increased, the ‘peak potential’ was gradually shifted towards more depolarized potentials, demon- strating that there was indeed a very good correlation between the ‘peak potential’ and the relative abundance of hERG1b. The results of the oocyte experiments are summarized in figure 3B. The ‘peak potentials’ were 242.661.1 mV (hERG1a; 0% hERG1b), 232.360.8 mV (20% hERG1b), 225.461.1 mV (40% hERG1b), 221.261.6 mV (60% hERG1b), 217.861.3 (80% hERG1b) and 213.860.6 mV (100% hERG1b) (P,0.05 for all comparisons except for 60% vs. 80% hERG1b and 80% vs. 100% hERG1b). When normalized to N there was no significant variation in the peak amplitude. The values were 1.1760.03 (hERG1a; 0% hERG1b), 1.1660.02 (20% hERG1b), 1.2160.03 (40% hERG1b), 1.2760.05 (60% hERG1b), 1.2960.04 (80% hERG1b) and 1.2760.03 (100% hERG1b). Macroscopic Kinetics of hERG1 Channels Are Dependent on the Relative Abundance of hERG1a and hERG1b Previously, it has been shown that hERG1a and hERG1b channels vary in kinetics of activation, deactivation and recovery from inactivation, i.e. the rates are increased by hERG1b, whereas no change in inactivation kinetics are observed.[8,9] To further verify our hypothesis we determined the kinetics of activation, deactivation and recovery from inactivation for hERG1 channels expressed in X. laevis oocytes using the same ratios of hERG1a to hERG1b as described above. In table S1 the time constants of these processes are given. Based on the average values, the relative changes in the time constants were calculated (table S1). Interestingly, linear correlations could be obtained for the relative changes in both tfast (r2 = 0.95), tslow (r2 = 0.99) and trec (r2 = 0.94) as a function of injected hERG1b cRNA (figure S1A-C). It was not possible to obtain a reliable estimate of trec for hERG1b channels alone due to a significant overlap with the transient capacitative current. Instead, the relative change in trec for hERG1b compared to hERG1a was calculated by extrapolation from the linear fit over the range 0–80% hERG1b (figure S1C). In contrast to tfast and trec the relative change in the time constant of activation was not linear. Rather, with the exception of 40% hERG1b the time constant was increased by approximately 1.5 fold for all ratios compared to hERG1a alone (figure S1D). For 40% hERG1b the time constant of activation was increased almost 2-fold. However, statistical analysis showed that although significantly different from hERG1a (0% hERG1b, P,0.05 for all comparisons) the time constants of activation for 20–100% hERG1b were not signifi- cantly different from each other. The possibility exists, that the intermediate kinetics observed in co-injected oocytes could arise from a channel population consisting of a mixture of distinct homomeric hERG1a and hERG1b channels. To address this issue, the observed tail currents recorded using the deactivation protocol from oocytes injected with 20–80% hERG1b were compared with the expected theoretical current decay assuming the formation of only homomeric channels. Averages of the observed current decay for Role of Cardiac hERG1 Isoforms Figure 3. The ‘peak potential’ is directly dependent on the relative abundance of hERG1 isoforms. A, Representative record- ings from X. laevis oocytes injected with different ratios of hERG1 isoforms. The same protocol as shown in figure 2A was used. Only currents recorded during the ramp are shown. For comparison, currents were normalized to the maximum peak current during the ramp and plotted against the command voltage. The dotted lines indicate the potential where the currents peaked (‘peak potential’) and are also used to indicate the isoform ratio (in % of hERG1b abundance). B, Summary data of the variation in ‘peak potential’. For comparison, the peak amplitude was normalized to N and plotted as a function of the ‘peak potential’. doi:10.1371/journal.pone.0009021.g003 20–80% hERG1b were calculated using the corresponding fitted time constants and their relative weights (table S1). The theoretical current decay was calculated as a weighted algebraic summation of the deactivation kinetics of hERG1a and hERG1b homomeric channels. Figure S2 shows the normalized decay of both the observed and theoretical situation at the four different isoform ratios. For all four comparisons, the (averaged) observed currents decay faster than the theoretical curves. Thus, the observed currents can not be explained by the presence of homomeric channels only. Similarly, the finding that the relative increase in activation kinetics did not change gradually as the relative abundance of hERG1b cRNA was increased also argues that the channel population upon co-expression of hERG1a and PLoS ONE | www.plosone.org 4 February 2010 | Volume 5 | Issue 2 | e9021