crispr-cas9-mediated-precise-knock-in-edits-in-zebrafish-hearts

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Simon Fraser University

Extracellular acidification regulates the biophysical properties of many voltage-gated potassium channels. Most often acidic pH reduces peak current and enhances current decay during depolarization. Here we review recent data from single channel and voltage clamp fluorimetry studies, which suggest that these two effects of protons are mediated by distinct kinetic processes. This new mechanistic insight directly demonstrates that whilst the enhanced decay of current observed with acidic pH is due to an accelerated entry of open channels into P/C-type inactivation, the main mechanism for the reduction in peak channel conductance is a stabilization of resting channels in closed-inactivated states. Thus acidic pH acts to reduce the mean burst time of conducting channels, as well as to prevent other channels from opening at all, and in so doing, reveals that both open- and closed-state inactivation processes can co-exist in K(V) channels.

Closed-state inactivation induced in K(V)1 channels by extracellular acidification.

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile(655) to Tyr(667) revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln(664) marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement. 

Proline scan of the HERG channel S6 helix reveals the location of the intracellular pore gate.

Zebrafish (Danio rerio) are widely used as vertebrate model in developmental genetics and functional genomics as well as in cardiac structure-function studies. The zebrafish heart has been increasingly used as a model of human cardiac function, in part, due to the similarities in heart rate and action potential duration and morphology with respect to humans. The teleostian zebrafish is in many ways a compelling model of human cardiac function due to the clarity afforded by its ease of genetic manipulation, the wealth of developmental biological information, and inherent suitability to a variety of experimental techniques. However, in addition to the numerous advantages of the zebrafish system are also caveats related to gene duplication (resulting in paralogs not present in human or other mammals) and fundamental differences in how zebrafish hearts function. In this review, we discuss the use of zebrafish as a cardiac function model through the use of techniques such as echocardiography, optical mapping, electrocardiography, molecular investigations of excitation-contraction coupling, and their physiological implications relative to that of the human heart. While some of these techniques (e.g., echocardiography) are particularly challenging in the zebrafish because of diminutive size of the heart (~1.5 mm in diameter) critical information can be derived from these approaches and are discussed in detail in this article.

The Zebrafish Heart as a Model of Mammalian Cardiac Function.

Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τ 34 ms, τ = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker.

KCNQ5 is a highly conserved gene encoding an important channel for neuronal function; it is widely expressed in the brain and generates M-type current. Exome sequencing identified de novo heterozygous missense mutations in four probands with intellectual disability, abnormal neurological findings, and treatment-resistant epilepsy (in two of four). Comprehensive analysis of this potassium channel for the four variants expressed in frog oocytes revealed shifts in the voltage dependence of activation, including altered activation and deactivation kinetics. Specifically, both loss-of-function and gain-of-function KCNQ5 mutations, associated with increased excitability and decreased repolarization reserve, lead to pathophysiology.

Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy.

Long QT syndrome (LQTS) is the most common cardiac ion channelopathy and has been found to be responsible for approximately 10% of sudden infant death syndrome (SIDS) cases. Despite increasing use of broad panels and now whole exome sequencing (WES) in the investigation of SIDS, the probability of identifying a pathogenic mutation in a SIDS victim is low. We report a family-based study who are afflicted by recurrent SIDS in which several members harbor a variant, p.Pro963Thr, in the C-terminal region of the human-ether-a-go-go (hERG) gene, published to be responsible for cases of LQTS type 2. Functional characterization was undertaken due to the variable phenotype in carriers, the discrepancy with published cases, and the importance of identifying a cause for recurrent deaths in a single family. Studies of the mutated ion channel in in vitro heterologous expression systems revealed that the mutation has no detectable impact on membrane surface expression, biophysical gating properties such as activation, deactivation and inactivation, or the amplitude of the protective current conducted by hERG channels during early repolarization. These observations suggest that the p.Pro963Thr mutation is not a monogenic disease-causing LQTS mutation despite evidence of co-segregation in two siblings affected by SIDS. Our findings demonstrate some of the potential pitfalls in post-mortem molecular testing and the importance of functional testing of gene variants in determining disease-causation, especially where the impacts of cascade screening can affect multiple generations.

Functional characterization of a novel hERG variant in a family with recurrent sudden infant death syndrome: Retracting a genetic diagnosis.

The hERG channel is a key player in repolarization of the cardiac action potential. Pharmacological blockade of hERG channels depletes the cardiac repolarization reserve, increasing the risk of cardiac arrhythmias. The promiscuous nature of drug interactions with hERG presents a therapeutic challenge for drug design and development. Despite considerable effort, the mechanisms of drug binding remain incompletely understood. One proposed mechanism is that high-affinity drug binding preferentially occurs when channels are in the inactivated state. However, this has been difficult to test, since inactivation is rapid in hERG and access to the drug binding site is limited by slower opening of the activation gate. Here, we have directly assessed the role of inactivation in cisparide and terfenadine drug binding in mutant (I663P) hERG channels where the activation gate is trapped-open. We firstly demonstrate the utility of this approach by showing that inactivation, ion selectivity and high affinity drug binding are preserved in I663P mutant channels. We then assess the role of inactivation by applying cisapride and terfenadine at different membrane voltages, which induce varying degrees of inactivation. We show that the extent of block does not correlate with the extent of inactivation. These data suggest that inactivation is not a major determinant of cisapride or terfenadine binding in hERG channels.

Investigating the state dependence of drug binding in hERG channels using a trapped-open channel phenotype.

hERG channels underlie the delayed-rectifier K channel current (I), which is crucial for membrane repolarization and therefore termination of the cardiac action potential. hERG channels display unusually slow deactivation gating, which contributes to a resurgent current upon repolarization and may protect against post-depolarization-induced arrhythmias. hERG channels also exhibit robust mode shift behavior, which reflects the energetic separation of activation and deactivation pathways due to voltage sensor relaxation into a stable activated state. The mechanism of relaxation is unknown and likely contributes to slow hERG channel deactivation. Here, we use extracellular acidification to probe the structural determinants of voltage sensor relaxation and its influence on the deactivation gating pathway. Using gating current recordings and voltage clamp fluorimetry measurements of voltage sensor domain dynamics, we show that voltage sensor relaxation is destabilized at pH 6.5, causing an ∼20-mV shift in the voltage dependence of deactivation. We show that the pH dependence of the resultant loss of mode shift behavior is similar to that of the deactivation kinetics acceleration, suggesting that voltage sensor relaxation correlates with slower pore gate closure. Neutralization of D509 in S3 also destabilizes the relaxed state of the voltage sensor, mimicking the effect of protons, suggesting that acidic residues on S3, which act as countercharges to S4 basic residues, are involved in stabilizing the relaxed state and slowing deactivation kinetics. Our findings identify the mechanistic determinants of voltage sensor relaxation and define the long-sought mechanism by which protons accelerate hERG deactivation.

Extracellular protons accelerate hERG channel deactivation by destabilizing voltage sensor relaxation.

The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, I, in the heart, which is essential for normal cardiac electrical activity and rhythm. Slow deactivation is one of the hallmark features of the unusual gating characteristics of hERG channels, and plays a crucial role in providing a robust current that aids repolarization of the cardiac action potential. As such, there is significant interest in elucidating the underlying mechanistic determinants of slow hERG channel deactivation. Recent work has shown that the hERG channel S4 voltage sensor is stabilized following activation in a process termed relaxation. Voltage sensor relaxation results in energetic separation of the activation and deactivation pathways, producing a hysteresis, which modulates the kinetics of deactivation gating. Despite widespread observation of relaxation behaviour in other voltage-gated K channels, such as , Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase -VSP, the relationship between stabilization of the activated voltage sensor by the open pore and voltage sensor relaxation in the control of deactivation has only recently begun to be explored. In this review, we discuss present knowledge and questions raised related to the voltage sensor relaxation mechanism in hERG channels and compare structure-function aspects of relaxation with those observed in related ion channels. We focus discussion, in particular, on the mechanism of coupling between voltage sensor relaxation and deactivation gating to highlight the insight that these studies provide into the control of hERG channel deactivation gating during their physiological functioning.

Modulation of hERG K Channel Deactivation by Voltage Sensor Relaxation.

Human ether-à-go-go related gene (hERG) K channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach. hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

The hERG channel activator, RPR260243, enhances protective  current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243.

Mathematical models of voltage-gated ion channels are used in basic research, industrial and clinical settings. These models range in complexity, but typically contain numerous variables representing the proportion of channels in a given state, and parameters describing the voltage-dependent rates of transition between states. An open problem is selecting the appropriate degree of complexity and structure for an ion channel model given data availability. Here, we simplify a model of the cardiac human Ether-à-go-go related gene (hERG) potassium ion channel, which carries cardiac , using the manifold boundary approximation method (MBAM). The MBAM approximates high-dimensional model-output manifolds by reduced models describing their boundaries, resulting in models with fewer parameters (and often variables). We produced a series of models of reducing complexity starting from an established five-state hERG model with 15 parameters. Models with up to three fewer states and eight fewer parameters were shown to retain much of the predictive capability of the full model and were validated using experimental hERG1a data collected in HEK293 cells at 37°C. The method provides a way to simplify complex models of ion channels that improves parameter identifiability and will aid in future model development.

Article	Year
Closed-state inactivation induced in K(V)1 channels by extracellular acidification. Channels (Austin, Tex.)\| PubMed ID: 18849651	2008
Proline scan of the HERG channel S6 helix reveals the location of the intracellular pore gate. Biophysical journal\| PubMed ID: 24606930	2014
The Zebrafish Heart as a Model of Mammalian Cardiac Function. Reviews of physiology, biochemistry and pharmacology\| PubMed ID: 27538987	2016
Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker. Biophysical journal\| PubMed ID: 28122216	2017
Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy. American journal of human genetics\| PubMed ID: 28669405	2017
Functional characterization of a novel hERG variant in a family with recurrent sudden infant death syndrome: Retracting a genetic diagnosis. Forensic science international\| PubMed ID: 29331839	2018
Investigating the state dependence of drug binding in hERG channels using a trapped-open channel phenotype. Scientific reports\| PubMed ID: 29563525	2018
Extracellular protons accelerate hERG channel deactivation by destabilizing voltage sensor relaxation. The Journal of general physiology\| PubMed ID: 30530765	2019
Modulation of hERG K Channel Deactivation by Voltage Sensor Relaxation. Frontiers in pharmacology\| PubMed ID: 32184724	2020
The hERG channel activator, RPR260243, enhances protective current early in the refractory period reducing arrhythmogenicity in zebrafish hearts. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 32559136	2020
Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243. The Journal of general physiology\| PubMed ID: 34398210	2021
Ion channel model reduction using manifold boundaries. Journal of the Royal Society, Interface\| PubMed ID: 35946166	2022

Thomas W. Claydon

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