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Abstract
IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.
Highlights
IntroductionThe IKr current is a primary contributor to the repolarization of the human cardiac muscle, a delayed rectifier potassium current conducted by the Kv11.1 ion channel (more commonly referred to as human ether-a-go-go-related gene, or hERG1) (Sanguinetti et al, 1995; Li et al, 1996)
The IKr current is a primary contributor to the repolarization of the human cardiac muscle, a delayed rectifier potassium current conducted by the Kv11.1 ion channel (Sanguinetti et al, 1995; Li et al, 1996)
The kinetic modeling in section Markov Kinetic Models to Describe hERG a- and b-Isoforms shows a profound difference in hERG1a and hERG1b deactivation rates, where the quantitatively fitted parameters to the data suggested that the hERG1b late deactivation rate is between 8 and 15 times faster than hERG1a
Summary
The IKr current is a primary contributor to the repolarization of the human cardiac muscle, a delayed rectifier potassium current conducted by the Kv11.1 ion channel (more commonly referred to as human ether-a-go-go-related gene, or hERG1) (Sanguinetti et al, 1995; Li et al, 1996). This gating behavior produces a resurgent current that peaks during the repolarization phase. Since the physiological role of IKr is to repolarize the late phase of cardiac action potentials, hERG1 has a clear link to these arrhythmias (Robertson et al, 2008; Gustina and Trudeau, 2009; Robertson, 2012; Vandenberg et al, 2012). If IKr is reduced, due to loss-of-function mutations or action of small molecules (drugs), patients are more likely to develop severe arrhythmias initiated by premature beats
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