Zebrafish cardiac repolarization does not functionally depend on the expression of the hERG1b-like transcript.

Zebrafish provide a translational model of human cardiac function. Their similar cardiac electrophysiology enables screening of human cardiac repolarization disorders, drug arrhythmogenicity, and novel antiarrhythmic therapeutics. However, while zebrafish cardiac repolarization is driven by delayed rectifier potassium channel current (IKr), the relative role of alternate channel transcripts is uncertain. While human ether-a-go-go-related-gene-1a (hERG1a) is the dominant transcript in humans, expression of the functionally distinct alternate transcript, hERG1b, modifies the electrophysiological and pharmacologic IKr phenotype. Studies of zebrafish IKr are frequently translated without consideration for the presence and impact of hERG1b in humans. Here, we performed phylogenetic analyses of all available KCNH genes from Actinopterygii (ray-finned fishes). Our findings confirmed zebrafish cardiac zkcnh6a as the paralog of human hERG1a (hKCNH2a), but also revealed evidence of a hERG1b (hKCNH2b)-like N-terminally truncated gene, zkcnh6b, in zebrafish. zkcnh6b is a teleost-specific variant that resulted from the 3R genome duplication. qRT-PCR showed dominant expression of zkcnh6a in zebrafish atrial and ventricular tissue, with low levels of zkcnh6b. Functional evaluation of zkcnh6b in a heterologous system showed no discernable function under the conditions tested, and no influence on zkcnh6a function during the zebrafish ventricular action potential. Our findings provide the first descriptions of the zkcnh6b gene, and show that, unlike in humans, zebrafish cardiac repolarization does not rely upon co-assembly of zERG1a/zERG1b. Given that hERG1b modifies IKr function and drug binding in humans, our findings highlight the need for consideration when translating hERG variant effects and toxicological screens in zebrafish, which lack a functional hERG1b-equivalent gene.

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

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.

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

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.NEW & NOTEWORTHY 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 Zebrafish Heart as a Model of Mammalian Cardiac Function.

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.

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

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 (τfast = 34 ms, τslow = 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.

hiPSC-derived cardiomyocytes as a model to study the role of small-conductance Ca2+-activated K+ (SK) ion channel variants associated with atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, has been associated with different electrophysiological, molecular, and structural alterations in atrial cardiomyocytes. Therefore, more studies are required to elucidate the genetic and molecular basis of AF. Various genome-wide association studies (GWAS) have strongly associated different single nucleotide polymorphisms (SNPs) with AF. One of these GWAS identified the rs13376333 risk SNP as the most significant one from the 1q21 chromosomal region. The rs13376333 risk SNP is intronic to the KCNN3 gene that encodes for small conductance calcium-activated potassium channels type 3 (SK3). However, the functional electrophysiological effects of this variant are not known. SK channels represent a unique family of K+ channels, primarily regulated by cytosolic Ca2+ concentration, and different studies support their critical role in the regulation of atrial excitability and consequently in the development of arrhythmias like AF. Since different studies have shown that both upregulation and downregulation of SK3 channels can lead to arrhythmias by different mechanisms, an important goal is to elucidate whether the rs13376333 risk SNP is a gain-of-function (GoF) or a loss-of-function (LoF) variant. A better understanding of the functional consequences associated with these SNPs could influence clinical practice guidelines by improving genotype-based risk stratification and personalized treatment. Although research using native human atrial cardiomyocytes and animal models has provided useful insights, each model has its limitations. Therefore, there is a critical need to develop a human-derived model that represents human physiology more accurately than existing animal models. In this context, research with human induced pluripotent stem cells (hiPSC) and subsequent generation of cardiomyocytes derived from hiPSC (hiPSC-CMs) has revealed the underlying causes of various cardiovascular diseases and identified treatment opportunities that were not possible using in vitro or in vivo studies with animal models. Thus, the ability to generate atrial cardiomyocytes and atrial tissue derived from hiPSCs from human/patients with specific genetic diseases, incorporating novel genetic editing tools to generate isogenic controls and organelle-specific reporters, and 3D bioprinting of atrial tissue could be essential to study AF pathophysiological mechanisms. In this review, we will first give an overview of SK-channel function, its role in atrial fibrillation and outline pathophysiological mechanisms of KCNN3 risk SNPs. We will then highlight the advantages of using the hiPSC-CM model to investigate SNPs associated with AF, while addressing limitations and best practices for rigorous hiPSC studies.

Continuous Positive Airway Pressure plus Mandibular Advancement Therapy (PAPMAT): study protocol for an adaptive randomised crossover trial comparing the benefits and costs of combining two established treatments for obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) involves repeated breathing pauses during sleep due to upper airway obstruction. It causes excessive daytime sleepiness and has other health impacts. Continuous positive airway pressure (CPAP) therapy is effective first line treatment for moderate to severe OSA. Unfortunately, many patients have difficulty tolerating CPAP and pressure intolerance is probably an important contributing factor. Mandibular advancement devices (MAD) are an alternative to CPAP. They are worn in the mouth during sleep to reduce airway obstruction. There is some evidence that, when used in combination with CPAP, MADs improve airway anatomy enough to reduce the CPAP pressure required to treat OSA and that this combination therapy could improve CPAP adherence. METHODS: Consecutive patients starting on CPAP for moderate to severe OSA will be recruited at a regional NHS sleep service. Patients with high CPAP pressure requirements after initial titration, who satisfy all entry criteria and consent to participate, will undertake a 2-arm randomised crossover trial. The arms will be (i) standalone CPAP and (ii) CPAP + MAD therapy. Each arm will last 12 weeks, including 2 weeks acclimatisation. CPAP machines will be auto-titrating and with facility for data download, so the impact of MAD on CPAP pressure requirements and CPAP adherence can be easily measured. The primary outcome will be CPAP adherence. Secondary outcomes will include measures of OSA severity, patient-reported outcome measures including subjective daytime sleepiness, quality of life, and treatment preference at the trial exit and health service use. Cost-effectiveness analyses will be undertaken. DISCUSSION: If the intervention is shown to be effective and cost-effective in improving adherence in this standard CPAP-eligible OSA patient population it would be relatively straightforward to introduce into existing OSA treatment pathways, within the wider NHS and more widely. Both MAD and CPAP are already used by sleep services so their combination would require only minor adjustments to existing clinical pathways. It would be straightforward to disseminate the results of the study through regional, national, and international respiratory meetings. The health economics analysis would provide cost-effectiveness data to inform service planning and clinical guidelines through policy briefing papers, including those by NICE and SIGN. TRIAL REGISTRATION: PAPMAT was registered with ISRCTN prior to recruitment beginning (ISRCTN Registry 2021): https://www.isrctn.com/ISRCTN33966032 . Registered on 17th November 2021.

Photothermal release of small molecules from gold nanoparticles in live cells.

The ability of gold (Au) nanoparticles (NPs) to generate heat efficiently by absorbing visible and near-infrared (NIR) light holds great promise as a means to trigger chemical and biochemical events near the NPs. Previous demonstrations show that pulsed laser irradiation can selectively elicit the release of a fluorescent dye covalently anchored to the NP surface through a heat-labile linker without measurably changing the temperature of the surroundings. This article reports that the authors demonstrate the biological efficacy of this approach to photodelivery by showing that the decorated Au NPs are rapidly internalized by cells, are stable under physiological conditions, are nontoxic, and exhibit nonlethal photorelease following exposure to pulsed laser radiation. These observations, further supported by the versatility of our delivery motif, reaffirm the potential for further development of nonlethal photothermal therapeutics and their future relevance to such fields as gene therapy and stem-cell differentiation.

Ion channel model reduction using manifold boundaries.

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 IKr, 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.

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 phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

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

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.

Modulation of hERG K+ Channel Deactivation by Voltage Sensor Relaxation.

The hERG (human-ether-à-go-go-related gene) channel underlies the rapid delayed rectifier current, Ikr, 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 Shaker, Kv1.2 and Kv3.1, as well as the voltage-sensing phosphatase Ci-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.

A direct demonstration of closed-state inactivation of K+ channels at low pH.

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker alpha-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K(+) or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at -80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

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

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.

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker.

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V((1/2))) values ranging from approximately -40 mV to approximately +30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes ("fast" and "slow"). "Slow" gating (tau(act) = 90 +/- 6 ms at +35 mV) was associated with a V((1/2)) of activation of +16.6 +/- 1.1 mV, whereas "fast" gating (tau(act) = 4.5 +/- 1.7 ms at +35 mV) was associated with a V((1/2)) of activation of -18.8 +/- 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate "fast" and "slow" activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished "slow" activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from "slow" to "fast", suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.

Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence.

The Kv1.2 channel, with its high resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at A291C reports two distinct rearrangements of the voltage sensor domains, and a comparative fluorescence scan of the S4 and S3-S4 linker residues in Shaker and Kv1.2 shows important differences in their emission at other homologous residues. Kv1.2 shows a rapid decrease in A291C emission with a time constant of 1.5 +/- 0.1 ms at 60 mV (n = 11) that correlates with gating currents and reports on translocation of the S4 and S3-S4 linker. However, unlike any Kv channel studied to date, this fast component is dwarfed by a larger, slower quenching of TMRM emission during depolarizations between -120 and -50 mV (tau = 21.4 +/- 2.1 ms at 60 mV, V(1/2) of -73.9 +/- 1.4 mV) that is not seen in either Shaker or Kv1.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltage-dependent manner that matches the time course of Kv1.2 ionic current deactivation. Fluorophores placed directly in S1 and S2 at I187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost when the extracellular S1-S2 linker of Kv1.2 is replaced with that of Kv1.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close.

Rapid outer pore movements after opening in a KV1 potassium channel are revealed by TMRM fluorescence from the S3-S4 linker, and modulated by extracellular potassium.

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker KV channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian KV1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of KV1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth, that is not seen in Shaker despite significant homology between it and KV1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and KV channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K+ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K+, and an IC50 of 29 mM K+o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K+, and further support the idea that outer pore motions occur rapidly after KV1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.

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

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.

Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel.

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3-S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 +/- 3% of the total signal and occurred with a tau of 3.4 +/- 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K(+) to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

4-aminopyridine prevents the conformational changes associated with p/c-type inactivation in shaker channels.

The effect of 4-aminopyridine (4-AP) on Kv channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation- and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 +/- 3 to 28 +/- 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 +/- 3 to 72 +/- 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization (tau reduced from 87 +/- 15 to 40 +/- 1 ms and from 739 +/- 83 to 291 +/- 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of Kv channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation.