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Characterization of cardiac hERG1 isoforms

Anders Peter Larsen

Summary

In the heart, the voltage-dependent delayed rectifier potassium current IK is responsible for repolarization of the cardiac action potential. The current can be separated into two components, the slowly activating delayed rectifier current (IKs) and the rapidly activating delayed rectifier current (IKr). The molecular correlate of IKr has been shown to be the ERG1 (ether-a-go-go related gene type 1) potassium channel encoded by the KCNH2 gene.
Mutations in KCNH2 have been associated with arrhythmic disorders such as the long QT (LQT) and the short QT (SQT) syndromes demonstrating the importance of this current in the repolarization of cardiac tissue. Furthermore, many drugs are known to bind to the ERG channel and inhibit the current resulting in an acquired form of the LQT syndrome. Accordingly, IKr and its molecular composition has been the focus of much research. Several isoforms of the ERG1 protein have been described. Traditionally, only the ERG1a isoform has been associated with cardiac IKr. Recently, it has been suggested that another isoform, ERG1b, also contributes to the generation of cardiac IKr. Structurally, ERG1a and ERG1b are similar, except that ERG1b has a shorter and unique N-terminus. However, the functional implications of ERG1b are largely unknown. The aim of this thesis was to characterize the human variant of the ERG1b isoform in terms of electrophysiological properties and functional interaction with hERG1a, and further, to investigate the potential implications of hERG1b on the properties of excitable cardiac cells. Electrophysiological characterization in a mammalian expression system showed that the homomeric hERG1a and hERG1b channels differ in their kinetics of activation, deactivation
and recovery from inactivation. For all three processes, hERG1b channels displayed faster kinetics. In contrast, the rate of inactivation was not significantly different between the two homomeric channels. Co-expression of hERG1a and hERG1b resulted in currents that were dominated by the properties of hERG1b.
Quantification of hERG1a and hERG1b mRNA from healthy human hearts showed that hERG1b on average constitutes 19 % in the right atrium and 12 % in the left ventricle of the total hERG1 mRNA. Co-expression of hERG1a and hERG1b in X. laevis oocytes showed that these ratios were indeed sufficient to affect the deactivation properties markedly.
A more detailed examination of the macroscopic currents resulting from co-expression of hERG1a and hERG1b in X. laevis oocytes revealed that the properties of the current is dependent on the relative abundance of the isoforms. Specifically, the results demonstrated 1) that the changes in macroscopic kinetics could only be explained by the formation of heteromeric channels, and 2) that changes in deactivation and recovery from inactivation were correlated to the relative abundance of hERG1b whereas activation was not. Slow activation kinetics and thus slow activation gating was observed only for hERG1a homomeric channels.
As a result of the differential changes in kinetics, more charge is effectively passed in the presence of hERG1b.
The observed differences in kinetic properties lead to the hypothesis that the heterogeneity of native IKr could be reproduced by differential expression of ERG1a and ERG1b isoforms.
Using a combination of heterologous expression experiments and computational simulations to elucidate the role of ERG1b in cardiac repolarization it was shown that the heterogeneity of native IKr could be reproduced by differential expression of ERG1a and ERG1b isoforms. Furthermore, the data suggested that subunit dependent kinetic changes, especially in kinetics of deactivation and recovery from inactivation, are important for the functional properties of ERG1 currents and hence cardiac IKr. Importantly, the simulations showed that differential expression of ERG1a and ERG1b may in turn modulate both action potential duration (APD) and the slope of the restitution curve.
In conclusion, the present work demonstrates that hERG1b is likely to play a role in the formation of the native IKr current. The properties of macroscopic hERG1 currents can be modulated by controlling the relative abundance of hERG1a and hERG1b, indicating that differential expression of ERG1 isoforms may serve as a physiological mechanism of tuning cellular excitability. Importantly, differential expression of ERG1 isoforms may contribute to the heterogeneity of IKr kinetics, APD and APD restitution observed in mammalian hearts.