Defining the Polycystin Pharmacophore Through HTS & Computational Biophysics.

Background And Purpose: Polycystins (PKD2, PKD2L1) are voltage-gated and Ca-modulated members of the transient receptor potential (TRP) family of ion channels. Loss of PKD2L1 expression results in seizure-susceptibility and autism-like features in mice, whereas variants in PKD2 cause autosomal dominant polycystic kidney disease. Despite decades of evidence clearly linking their dysfunction to human disease and demonstrating their physiological importance in the brain and kidneys, the polycystin pharmacophore remains undefined.

Transient titin-dependent ventricular defects during development lead to adult atrial arrhythmia and impaired contractility.

Developmental causes of the most common arrhythmia, atrial fibrillation (AF), are poorly defined, with compensation potentially masking arrhythmic risk. Here, we delete 9 amino acids (Δ9) within a conserved domain of the giant protein titin's A-band in zebrafish and human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). We find that zebrafish embryos' cardiac morphology is perturbed and accompanied by reduced functional output, but ventricular function recovers within days.

Molecular and cellular context influences SCN8A variant function.

Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.

Independent compartmentalization of functional, metabolic, and transcriptional maturation of hiPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components.

Expanded clinical phenotype spectrum correlates with variant function in SCN2A-related disorders.

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel Nav1.2 are rare, with clinically heterogeneous expressions that include epilepsy, autism and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relationship to channel function, 81 patients (36 female, 44%, median age 5.

Plural molecular and cellular mechanisms of pore domain encephalopathy.

variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path.

Engineered cocultures of iPSC-derived atrial cardiomyocytes and atrial fibroblasts for modeling atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia treatable with antiarrhythmic drugs; however, patient responses remain highly variable. Human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are useful for discovering precision therapeutics, but current platforms yield phenotypically immature cells and are not easily scalable for high-throughput screening. Here, primary adult atrial, but not ventricular, fibroblasts induced greater functional iPSC-aCM maturation, partly through connexin-40 and ephrin-B1 signaling.

Molecular and Cellular Context Influences SCN8A Variant Function.

Pathogenic variants in , which encodes the voltage-gated sodium (Na ) channel Na 1.6, are associated with neurodevelopmental disorders including epileptic encephalopathy. Previous approaches to determine variant function may be confounded by the use of a neonatal-expressed alternatively spliced isoform of Na 1.

Epilepsy-associated SCN2A (NaV1.2) variants exhibit diverse and complex functional properties.

Pathogenic variants in voltage-gated sodium (NaV) channel genes including SCN2A, encoding NaV1.2, are discovered frequently in neurodevelopmental disorders with or without epilepsy. SCN2A is also a high-confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID).

Epilepsy-associated (Na 1.2) Variants Exhibit Diverse and Complex Functional Properties.

Pathogenic variants in neuronal voltage-gated sodium (Na ) channel genes including , which encodes Na 1.2, are frequently discovered in neurodevelopmental disorders with and without epilepsy. is also a high confidence risk gene for autism spectrum disorder (ASD) and nonsyndromic intellectual disability (ID).

Cholesterol-induced suppression of Kir2 channels is mediated by decoupling at the inter-subunit interfaces.

Cholesterol is a major regulator of multiple types of ion channels. Although there is increasing information about cholesterol binding sites, the molecular mechanisms through which cholesterol binding alters channel function are virtually unknown. In this study, we used a combination of Martini coarse-grained simulations, a network theory-based analysis, and electrophysiology to determine the effect of cholesterol on the dynamic structure of the Kir2.

Predicting the functional impact of KCNQ1 variants with artificial neural networks.

Recent advances in experimental and computational protein structure determination have provided access to high-quality structures for most human proteins and mutants thereof. However, linking changes in structure in protein mutants to functional impact remains an active area of method development. If successful, such methods can ultimately assist physicians in taking appropriate treatment decisions.

High-throughput evaluation of epilepsy-associated KCNQ2 variants reveals functional and pharmacological heterogeneity.

Hundreds of genetic variants in KCNQ2 encoding the voltage-gated potassium channel KV7.2 are associated with early onset epilepsy and/or developmental disability, but the functional consequences of most variants are unknown. Absent functional annotation for KCNQ2 variants hinders identification of individuals who may benefit from emerging precision therapies.

Genomic Autopsy of Sudden Deaths in Young Individuals.

Importance: Postmortem genetic testing of young individuals with sudden death has previously identified pathogenic gene variants. However, prior studies primarily considered highly penetrant monogenic variants, often without detailed decedent and family clinical information.

Objective: To assess genotype and phenotype risk in a diverse cohort of young decedents with sudden death and their families.

Functional evaluation of human ion channel variants using automated electrophysiology.

Patch clamp recording enabled a revolution in cellular electrophysiology, and is useful for evaluating the functional consequences of ion channel gene mutations or variants associated with human disorders called channelopathies. However, due to massive growth of genetic testing in medical practice and research, the number of known ion channel variants has exploded into the thousands. Fortunately, automated methods for performing patch clamp recording have emerged as important tools to address the explosion in ion channel variants.

Targeting the Microtubule EB1-CLASP2 Complex Modulates Na1.5 at Intercalated Discs.

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Disease-linked supertrafficking of a potassium channel.

Gain-of-function (GOF) mutations in the voltage-gated potassium channel subfamily Q member 1 (KCNQ1) can induce cardiac arrhythmia. In this study, it was tested whether any of the known human GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface-"supertrafficking." Seven of the 15 GOF mutants tested were seen to surface traffic more efficiently than the WT channel.

Dyshomeostatic modulation of Ca-activated K channels in a human neuronal model of KCNQ2 encephalopathy.

Mutations in , which encodes a pore-forming K channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons.

Allosteric mechanism for KCNE1 modulation of KCNQ1 potassium channel activation.

The function of the voltage-gated KCNQ1 potassium channel is regulated by co-assembly with KCNE auxiliary subunits. KCNQ1-KCNE1 channels generate the slow delayed rectifier current, I, which contributes to the repolarization phase of the cardiac action potential. A three amino acid motif (F57-T58-L59, FTL) in KCNE1 is essential for slow activation of KCNQ1-KCNE1 channels.

Publisher Correction: Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome.

The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (K) channels KCNQ1 and hERG as well as the voltage-gated sodium (Na) channel encoded by . Each channel performs a highly distinct function, despite sharing a common topology and structural components.

Striatal Kir2 K+ channel inhibition mediates the antidyskinetic effects of amantadine.

Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying K+ channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs) - not NMDA receptors.

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin.