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 α-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

BSP implementation of the 2017 classification of periodontal diseases: a practice retrospective

Introduction The new world classification of periodontal diseases and conditions was developed in 2017. The British Society of Periodontology and Implant Dentistry (BSP) implemented the classification in a series of papers published in the British Dental Journal in January 2019. Aims and objectives This study aimed to investigate if the BSP implementation was workable in general dental practice and to reveal if any lessons were learnt from its regular use two years following its release. Materials and methods This was a cross-sectional, retrospective, non-intervention analysis of a patient population (n = 891) drawn from a complete list of a private dental surgeon. Diagnostic and demographic data were drawn from the patient records, collated and analysed using SPSS Statistics v26. Results Diagnoses derived from the new classification were identified for 92% of subjects, indicating a high level of implementation. In total, 20.9% of subjects were diagnosed with periodontitis, and of these, 57% were unstable, 39% stable and 4% in remission. The mean bleeding on probing score across the cohort was 7.7%. Moreover, 76% of the non-periodontitis patients were diagnosed with 'clinical gingival health', 23% with localised gingivitis and 1% with generalised gingivitis. Conclusion The new classification has been found to be readily implemented in a general practice setting. Use of the new classification allows for close monitoring of periodontal status, and as a result, close monitoring of the effectiveness of pathways of care

The use of non-surgical interventions in patients with periimplantitis; a systematic review and meta-analysis

Objectives: To systematically assess the efficacy of different non‐surgical treatment methods to manage peri‐implantitis reported to date in the literature, together with its correlation with time following therapy. Materials and Methods: A systematic literature review was undertaken to identify randomised control trials of the non‐surgical management of peri‐implantitis published up to November 2019. The search was limited to English language human studies containing follow‐up periods of ≥3 months and for sample size of 10 or more patients. A meta‐analysis was implemented for the following clinical parameters: Peri‐implant pocket depth (PPD), bleeding on probing (BOP), clinical attachment level (CAL), radiographic bone loss (RBL) and mucosal recession (MR). Results: Twelve articles met the inclusion criteria. Two principal treatment modalities were identified; mechanical debridement and laser therapy, with two adjunctive therapies antimicrobial and antiseptic agents. Non‐surgical interventions (ultrasonic scalers, Er:YAG laser and powdered air‐abrasive devices) showed significant clinical improvement in the short term (<3 months). Clinical benefit was demonstrated with the adjunctive use of antimicrobial agents in the short term but diminished with time. Antiseptic agents alone have no significant effect. Non‐surgical therapies applied in these studies failed to arrest mucosal recession, peri‐implant bone loss or reduce the counts of viable pathogens in the long term. Conclusion: The evidence demonstrate that the clinical parameters of peri‐implantitis, i.e. BOP, PPD and CAL may all be improved by simple mechanical debridement, using either ultrasonic instrumentation or Er:YAG laser therapy; adjunctive antimicrobial and antiseptic therapy. Further randomised control trials in this area are, however, required

Surgical therapy for peri-implantitis management: a systematic review and meta-analysis

Aim Peri‐implantitis is a common cause of late implant failure. Studies have investigated different treatment strategies. The effectiveness of these modalities, however, remains unclear. This study aimed to evaluate the success of surgical peri‐implantitis treatment using clinical and radiographic parameters. Material and methods A systematic review of published literature was employed. Key words were selected to conduct an electronic search using four databases for literature on human clinical studies. Meta‐analyses were carried out for clinical probing, pocket depth and radiographic bone level. Results A total of 16 papers met the inclusion criteria. Four treatment modalities to supplement mechanical debridement were identified: (i) apically repositioned flap, (ii) chemical surface decontamination, (iii) implantoplasty, and (iv) bone augmentation. Inconsistent results were evident which were dependent on several treatment‐independent factors. No clinical benefits were identified for the additional use of surface decontamination, while limited evidence demonstrated improvement of clinical and radiographic outcomes after implantoplasty. The effect of bone augmentation appeared limited to ‘filling’ radiographic defects. Conclusions The outcomes of the currently available surgical interventions for peri‐implantitis remain unpredictable. There is no reliable evidence to suggest which methods are the most effective. Further randomised‐controlled studies are needed to identify the best treatment methods

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 5-state hERG model with 15 parameters. Models with up to 3 fewer states and 8 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

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

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

Consensus statement on abusive head trauma in infants and young children

Abusive head trauma (AHT) is the leading cause of fatal head injuries in children younger than 2 years. A multidisciplinary team bases this diagnosis on history, physical examination, imaging and laboratory findings. Because the etiology of the injury is multifactorial (shaking, shaking and impact, impact, etc.) the current best and inclusive term is AHT. There is no controversy concerning the medical validity of the existence of AHT, with multiple components including subdural hematoma, intracranial and spinal changes, complex retinal hemorrhages, and rib and other fractures that are inconsistent with the provided mechanism of trauma. The workup must exclude medical diseases that can mimic AHT. However, the courtroom has become a forum for speculative theories that cannot be reconciled with generally accepted medical literature. There is no reliable medical evidence that the following processes are causative in the constellation of injuries of AHT: cerebral sinovenous thrombosis, hypoxic-ischemic injury, lumbar puncture or dysphagic choking/vomiting. There is no substantiation, at a time remote from birth, that an asymptomatic birth-related subdural hemorrhage can result in rebleeding and sudden collapse. Further, a diagnosis of AHT is a medical conclusion, not a legal determination of the intent of the perpetrator or a diagnosis of murder. We hope that this consensus document reduces confusion by recommending to judges and jurors the tools necessary to distinguish genuine evidence-based opinions of the relevant medical community from legal arguments or etiological speculations that are unwarranted by the clinical findings, medical evidence and evidence-based literature