MiR-21-5p alleviates trigeminal neuralgia in rats through down-regulation of voltage-gated potassium channel Kv1.1. / MiR-21-5p通过下调电压门控钾离子通道Kv1.1è¡¨è¾¾å‡è½»å¤§é¼ ä¸‰å‰ç¥žç»ç—›.

OBJECTIVES: Trigeminal neuralgia (TN) is a common neuropathic pain. Voltage-gated potassium channel (Kv) has been confirmed to be involved in the occurrence and development of TN, but the specific mechanism is still unclear. MicroRNA may be involved in neuropathic pain by regulating the expression of Kv channels and neuronal excitability in trigeminal ganglion (TG). This study aims to explore the relationship between Kv1.1 and miR-21-5p in TG with a TN model, evaluate whether miR-21-5p has a regulatory effect on Kv1.1, and to provide a new target and experimental basis for the treatment of TN. METHODS: A total of 48 SD rats were randomly divided into 6 groups: 1) a sham group (n=12), the rats were only sutured at the surgical incision without nerve ligation; 2) a sham+agomir NC group (n=6), the sham rats were microinjected with agomir NC through stereotactic brain injection in the surgical side of TG; 3) a sham+miR-21-5p agomir group (n=6), the sham rats were microinjected with miR-21-5p agomir via stereotactic brain injection in the surgical side of TG; 4) a TN group (n=12), a TN rat model was constructed using the chronic constriction injury of the distal infraorbital nerve (dIoN-CCI) method with chromium intestinal thread; 5) a TN+antagonist NC group (n=6), TN rats were microinjected with antagonist NC through stereotactic brain injection method in the surgical side of TG; 6) a TN+miR-21-5p antagonist group (n=6), TN rats were microinjected with miR-21-5p antagonist through stereotactic brain injection in the surgical side of TG. The change of mechanical pain threshold in rats of each group after surgery was detected. The expressions of Kv1.1 and miR-21-5p in the operative TG of rats were detected by Western blotting and real-time reverse transcription polymerase chain reaction. Dual luciferase reporter genes were used to determine whether there was a target relationship between Kv1.1 and miR-21-5p and whether miR-21-5p directly affected the 3'-UTR terminal of KCNA1. The effect of brain stereotaxic injection was evaluated by immunofluorescence assay, and then the analogue of miR-21-5p (agomir) and agomir NC were injected into the TG of rats in the sham group by brain stereotaxic apparatus to overexpress miR-21-5p. The miR-21-5p inhibitor (antagomir) and antagomir NC were injected into TG of rats in the TN group to inhibit the expression of miR-21-5p. The behavioral changes of rats before and after administration were observed, and the expression changes of miR-21-5p and Kv1.1 in TG of rats after intervention were detected. RESULTS: Compared with the baseline pain threshold, the facial mechanical pain threshold of rats in the TN group was significantly decreased from the 5th to 15th day after the surgery (P<0.05), and the facial mechanical pain threshold of rats in the sham group was stable at the normal level, which proved that the dIoN-CCI model was successfully constructed. Compared with the sham group, the expression of Kv1.1 mRNA and protein in TG of the TN group was down-regulated (both P<0.05), and the expression of miR-21-5p was up-regulated (P<0.05). The results of dual luciferase report showed that the luciferase activity of rno-miR-21-5p mimics and KCNA1 WT transfected with 6 nmol/L or 20 nmol/L were significantly decreased compared with those transfected with mimic NC and wild-type KCNA1 WT, respectively (P<0.001). Compared with low dose rno-miR-21-5p mimics (6 nmol/L) co-transfection group, the relative activity of luciferase in the high dose rno-miR-21-5p mimics (20 nmol/L) cotransfection group was significantly decreased (P<0.001). The results of immunofluorescence showed that drugs were accurately injected into TG through stereotaxic brain. After the expression of miR-21-5p in the TN group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were increased. After overexpression of miR-21-5p in the sham group, the mechanical pain threshold and the expression of Kv1.1 mRNA and protein in TG were decreased. CONCLUSIONS: Both Kv1.1 and miR-21-5p are involved in TN and miR-21-5p can regulate Kv1.1 expression by binding to the 3'-UTR of KCNA1.

Desflurane improves electrical activity of neurons and alleviates oxygen-glucose deprivation-induced neuronal injury by activating the Kcna1-dependent Kv1.1 channel.

Several volatile anesthetics have presented neuroprotective functions in ischemic injury. This study investigates the effect of desflurane (Des) on neurons following oxygen-glucose deprivation (OGD) challenge and explores the underpinning mechanism. Mouse neurons HT22 were subjected to OGD, which significantly reduced cell viability, increased lactate dehydrogenase release, and promoted cell apoptosis. In addition, the OGD condition increased oxidative stress in HT22 cells, as manifested by increased ROS and MDA contents, decreased SOD activity and GSH/GSSG ratio, and reduced nuclear protein level of Nrf2. Notably, the oxidative stress and neuronal apoptosis were substantially blocked by Des treatment. Bioinformatics suggested potassium voltage-gated channel subfamily A member 1 (Kcna1) as a target of Des. Indeed, the Kcna1 expression in HT22 cells was decreased by OGD but restored by Des treatment. Artificial knockdown of Kcna1 negated the neuroprotective effects of Des. By upregulating Kcna1, Des activated the Kv1.1 channel, therefore enhancing K+ currents and inducing neuronal repolarization. Pharmacological inhibition of the Kv1.1 channel reversed the protective effects of Des against OGD-induced injury. Collectively, this study demonstrates that Des improves electrical activity of neurons and alleviates OGD-induced neuronal injury by activating the Kcna1-dependent Kv1.1 channel.

Basket to Purkinje Cell Inhibitory Ephaptic Coupling Is Abolished in Episodic Ataxia Type 1.

Dominantly inherited missense mutations of the KCNA1 gene, which encodes the KV1.1 potassium channel subunit, cause Episodic Ataxia type 1 (EA1). Although the cerebellar incoordination is thought to arise from abnormal Purkinje cell output, the underlying functional deficit remains unclear. Here we examine synaptic and non-synaptic inhibition of Purkinje cells by cerebellar basket cells in an adult mouse model of EA1. The synaptic function of basket cell terminals was unaffected, despite their intense enrichment for KV1.1-containing channels. In turn, the phase response curve quantifying the influence of basket cell input on Purkine cell output was maintained. However, ultra-fast non-synaptic ephaptic coupling, which occurs in the cerebellar 'pinceau' formation surrounding the axon initial segment of Purkinje cells, was profoundly reduced in EA1 mice in comparison with their wild type littermates. The altered temporal profile of basket cell inhibition of Purkinje cells underlines the importance of Kv1.1 channels for this form of signalling, and may contribute to the clinical phenotype of EA1.

Native American ataxia medicines rescue ataxia-linked mutant potassium channel activity via binding to the voltage sensing domain.

There are currently no drugs known to rescue the function of Kv1.1 voltage-gated potassium channels carrying loss-of-function sequence variants underlying the inherited movement disorder, Episodic Ataxia 1 (EA1). The Kwakwaka'wakw First Nations of the Pacific Northwest Coast used Fucus gardneri (bladderwrack kelp), Physocarpus capitatus (Pacific ninebark) and Urtica dioica (common nettle) to treat locomotor ataxia. Here, we show that extracts of these plants enhance wild-type Kv1.1 current, especially at subthreshold potentials. Screening of their constituents revealed that gallic acid and tannic acid similarly augment wild-type Kv1.1 current, with submicromolar potency. Crucially, the extracts and their constituents also enhance activity of Kv1.1 channels containing EA1-linked sequence variants. Molecular dynamics simulations reveal that gallic acid augments Kv1.1 activity via a small-molecule binding site in the extracellular S1-S2 linker. Thus, traditional Native American ataxia treatments utilize a molecular mechanistic foundation that can inform small-molecule approaches to therapeutically correcting EA1 and potentially other Kv1.1-linked channelopathies.

Novel Genetic Variants Expand the Functional, Molecular, and Pathological Diversity of KCNA1 Channelopathy.

The KCNA1 gene encodes Kv1.1 voltage-gated potassium channel α subunits, which are crucial for maintaining healthy neuronal firing and preventing hyperexcitability. Mutations in the KCNA1 gene can cause several neurological diseases and symptoms, such as episodic ataxia type 1 (EA1) and epilepsy, which may occur alone or in combination, making it challenging to establish simple genotype-phenotype correlations. Previous analyses of human KCNA1 variants have shown that epilepsy-linked mutations tend to cluster in regions critical for the channel's pore, whereas EA1-associated mutations are evenly distributed across the length of the protein. In this review, we examine 17 recently discovered pathogenic or likely pathogenic KCNA1 variants to gain new insights into the molecular genetic basis of KCNA1 channelopathy. We provide the first systematic breakdown of disease rates for KCNA1 variants in different protein domains, uncovering potential location biases that influence genotype-phenotype correlations. Our examination of the new mutations strengthens the proposed link between the pore region and epilepsy and reveals new connections between epilepsy-related variants, genetic modifiers, and respiratory dysfunction. Additionally, the new variants include the first two gain-of-function mutations ever discovered for KCNA1, the first frameshift mutation, and the first mutations located in the cytoplasmic N-terminal domain, broadening the functional and molecular scope of KCNA1 channelopathy. Moreover, the recently identified variants highlight emerging links between KCNA1 and musculoskeletal abnormalities and nystagmus, conditions not typically associated with KCNA1. These findings improve our understanding of KCNA1 channelopathy and promise to enhance personalized diagnosis and treatment for individuals with KCNA1-linked disorders.

kcna1a mutant zebrafish model episodic ataxia type 1 (EA1) with epilepsy and show response to first-line therapy carbamazepine.

OBJECTIVE: KCNA1 mutations are associated with a rare neurological movement disorder known as episodic ataxia type 1 (EA1), and epilepsy is a common comorbidity. Current medications provide only partial relief for ataxia and/or seizures, making new drugs needed. Here, we characterized zebrafish kcna1a-/- as a model of EA1 with epilepsy and compared the efficacy of the first-line therapy carbamazepine in kcna1a-/- zebrafish to Kcna1-/- rodents. METHODS: CRISPR/Cas9 mutagenesis was used to introduce a mutation in the sixth transmembrane segment of the zebrafish Kcna1 protein. Behavioral and electrophysiological assays were performed on kcna1a-/- larvae to assess ataxia- and epilepsy-related phenotypes. Real-time quantitative polymerase chain reaction (qPCR) was conducted to measure mRNA levels of brain hyperexcitability markers in kcna1a-/- larvae, followed by bioenergetics profiling to evaluate metabolic function. Drug efficacies were tested using behavioral and electrophysiological assessments, as well as seizure frequency in kcna1a-/- zebrafish and Kcna1-/- mice, respectively. RESULTS: Zebrafish kcna1a-/- larvae showed uncoordinated movements and locomotor deficits, along with scoliosis and increased mortality. The mutants also exhibited impaired startle responses when exposed to light-dark flashes and acoustic stimulation as well as hyperexcitability as measured by extracellular field recordings and upregulated fosab transcripts. Neural vglut2a and gad1b transcript levels were disrupted in kcna1a-/- larvae, indicative of a neuronal excitatory/inhibitory imbalance, as well as a significant reduction in cellular respiration in kcna1a-/- , consistent with dysregulation of neurometabolism. Notably, carbamazepine suppressed the impaired startle response and brain hyperexcitability in kcna1a-/- zebrafish but had no effect on the seizure frequency in Kcna1-/- mice, suggesting that this EA1 zebrafish model might better translate to humans than rodents. SIGNIFICANCE: We conclude that zebrafish kcna1a-/- show ataxia and epilepsy-related phenotypes and are responsive to carbamazepine treatment, consistent with EA1 patients. These findings suggest that kcna1-/- zebrafish are a useful model for drug screening as well as studying the underlying disease biology.

Circ_0000029 Interacts with the miR-576-5p/KCNA1 Axis to Hamper the Development of Pediatric Asthma in an Asthma-Like in vitro Assessment.

OBJECTIVE: Numerous circular RNAs (circRNAs) have been verified to execute crucial roles in "asthma-like" progression of the airway smooth muscle cells (ASMCs). The present study aimed to scrutinize the function and mechanism of circ_0000029 in pediatric asthma etiology in vitro. METHODS: A cell model of asthma was developed using ASMCs induced by platelet-derived growth factor BB (PDGF-BB). Western blotting and qRT-PCR were performed to determine the expression levels of circ_0000029, miR-576-5p, and KCNA1 in PDGF-BB-treated ASMCs. Dual-luciferase reporter, RNA-binding protein immunoprecipitation, and RNA pull-down experiments were conducted to validate targeting relationships. CCK-8 and Transwell assays were performed to evaluate the proliferative and migratory potential of ASMCs. The rate of apoptosis was analyzed using flow cytometry. RESULTS: Pronounced circ_0000029 and KCNA1 downregulation and high levels of miR-576-5p were observed in PDGF-BB-treated ASMCs. Circ_0000029 targets miR-576-5p to regulate KCNA1 expression. The loss of KCNA1 and upregulation of miR-576-5p dramatically impeded apoptosis but promoted ASMC migration and proliferation. Ectopic expression of circ_0000029 manifested the opposite outcome among ASMCs. Furthermore, KCNA1 deficiency and miR-576-5p upregulation counteracted the effects of circ_0000029 overexpression on ASMCs. CONCLUSIONS: Circ_0000029 represses the abnormal migration and growth of ASMCs by mediating miR-576-5p and KCNA1 expression levels. This suggests that the regulatory axis circ_0000029/miR-576-5p/KCNA1 is a potential target for pediatric asthma treatment.

KCNA1 gain-of-function epileptic encephalopathy treated with 4-aminopyridine.

Precision medicine for Mendelian epilepsy is rapidly developing. We describe an early infant with severely pharmacoresistant multifocal epilepsy. Exome sequencing revealed the de novo variant p.(Leu296Phe) in the gene KCNA1, encoding the voltage-gated K+ channel subunit KV 1.1. So far, loss-of-function variants in KCNA1 have been associated with episodic ataxia type 1 or epilepsy. Functional studies of the mutated subunit in oocytes revealed a gain-of-function caused by a hyperpolarizing shift of voltage dependence. Leu296Phe channels are sensitive to block by 4-aminopyridine. Clinical use of 4-aminopyridine was associated with reduced seizure burden, enabled simplification of co-medication and prevented rehospitalization.

Episodic Ataxia Type 1: Natural History and Effect on Quality of Life.

Episodic ataxia type 1 (EA1) is a rare autosomal potassium channelopathy, due to mutations in KCNA1. Patients have childhood onset of intermittent attacks of ataxia, dizziness or imbalance. In order to quantify the natural history of EA1, its effect on quality of life and in preparation for future clinical trials, we set up an international multi-centre study of EA1. We recruited thirty-three participants with EA1: twenty-three completed 1-year follow-up and eighteen completed 2-year follow-up. There was very little accumulation of disability or impairment over the course of the 2 years of the study. The outcome measures of ataxia (SARA and functional rating of ataxia) and the activities of daily living scale were largely stable over time. Self-reported health-related quality of life (SF-36) scores were lower across all domains than controls, in keeping with a chronic condition. Physical subdomain scores appeared to deteriorate over time, which seems to be driven by the female participants in the study. This is an interesting finding and warrants further study. Attacks of EA1 reported by participants in real time via an interactive voice response system showed that symptoms were not stereotyped; however, attack duration and frequency was stable between individuals. This large prospective study is the first ever completed in subjects with EA1. We document the natural history of the disorder over 2 years. These data will enable the development of outcome measures for clinical trials of treatment.

Radiochemical Synthesis and Evaluation of 3-[11C]Methyl-4-aminopyridine in Rodents and Nonhuman Primates for Imaging Potassium Channels in the CNS.

Demyelination, the loss of the insulating sheath of neurons, causes failed or slowed neuronal conduction and contributes to the neurological symptoms in multiple sclerosis, traumatic brain and spinal cord injuries, stroke, and dementia. In demyelinated neurons, the axonal potassium channels Kv1.1 and Kv1.2, generally under the myelin sheath, become exposed and upregulated. Therefore, imaging these channels using positron emission tomography can provide valuable information for disease diagnosis and monitoring. Here, we describe a novel tracer for Kv1 channels, [11C]3-methyl-4-aminopyridine ([11C]3Me4AP). [11C]3Me4AP was efficiently synthesized via Pd(0)-Cu(I) comediated Stille cross-coupling of a stannyl precursor containing a free amino group. Evaluation of its imaging properties in rats and nonhuman primates showed that [11C]3Me4AP has a moderate brain permeability and slow kinetics. Additional evaluation in monkeys showed that the tracer is metabolically stable and that a one-tissue compartment model can accurately model the regional brain time-activity curves. Compared to the related tracers [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) and [11C]3-methoxy-4-aminopyridine ([11C]3MeO4AP), [11C]3Me4AP shows lower initial brain uptake, which indicates reduced permeability to the blood-brain barrier and slower kinetics, suggesting higher binding affinity consistent with in vitro studies. While the slow kinetics and strong binding affinity resulted in a tracer with less favorable properties for imaging the brain than its predecessors, these properties may make 3Me4AP useful as a therapeutic.

Loss of functional System x-c uncouples aberrant postnatal neurogenesis from epileptogenesis in the hippocampus of Kcna1-KO mice.

Mutations in Kv1.1 (Kcna1) voltage-gated potassium channels in humans and mice generate network hyperexcitability, enhancing aberrant postnatal neurogenesis in the dentate subgranular zone, resulting in epilepsy and hippocampal hypertrophy. While Kcna1 loss stimulates proliferation of progenitor cell subpopulations, the identity of extrinsic molecular triggers linking network hyperexcitability to aberrant postnatal neurogenesis remains incomplete. System x-c (Sxc) is an inducible glutamate/cysteine antiporter that regulates extracellular glutamate. Here, we find that the functional unit of Sxc, xCT (Slc7a11), is upregulated in regions of Kcna1 knockout (KO) hippocampus, suggesting a contribution to both hyperplasia and epilepsy. However, Slc7a11 KO suppressed and rescued hippocampal enlargement without altering seizure severity in Kcna1-Slc7a11-KO mice. Microglial activation, but not astrocytosis, was also reduced. Our study identifies Sxc-mediated glutamate homeostasis as an essential non-synaptic trigger coupling aberrant postnatal neurogenesis and neuroimmune crosstalk, revealing that neurogenesis and epileptogenesis in the dentate gyrus are not mutually contingent events.

On-demand cell-autonomous gene therapy for brain circuit disorders.

Several neurodevelopmental and neuropsychiatric disorders are characterized by intermittent episodes of pathological activity. Although genetic therapies offer the ability to modulate neuronal excitability, a limiting factor is that they do not discriminate between neurons involved in circuit pathologies and "healthy" surrounding or intermingled neurons. We describe a gene therapy strategy that down-regulates the excitability of overactive neurons in closed loop, which we tested in models of epilepsy. We used an immediate early gene promoter to drive the expression of Kv1.1 potassium channels specifically in hyperactive neurons, and only for as long as they exhibit abnormal activity. Neuronal excitability was reduced by seizure-related activity, leading to a persistent antiepileptic effect without interfering with normal behaviors. Activity-dependent gene therapy is a promising on-demand cell-autonomous treatment for brain circuit disorders.

Artificial pore blocker acts specifically on voltage-gated potassium channel isoform KV1.6.

Among voltage-gated potassium channel (KV) isoforms, KV1.6 is one of the most widespread in the nervous system. However, there are little data concerning its physiological significance, in part due to the scarcity of specific ligands. The known high-affinity ligands of KV1.6 lack selectivity, and conversely, its selective ligands show low affinity. Here, we present a designer peptide with both high affinity and selectivity to KV1.6. Previously, we have demonstrated that KV isoform-selective peptides can be constructed based on the simplistic α-hairpinin scaffold, and we obtained a number of artificial Tk-hefu peptides showing selective blockage of KV1.3 in the submicromolar range. We have now proposed amino acid substitutions to enhance their activity. As a result, we have been able to produce Tk-hefu-11 that shows an EC50 of ≈70 nM against KV1.3. Quite surprisingly, Tk-hefu-11 turns out to block KV1.6 with even higher potency, presenting an EC50 of ≈10 nM. Furthermore, we have solved the peptide structure and used molecular dynamics to investigate the determinants of selective interactions between artificial α-hairpinins and KV channels to explain the dramatic increase in KV1.6 affinity. Since KV1.3 is not highly expressed in the nervous system, we hope that Tk-hefu-11 will be useful in studies of KV1.6 and its functions.

Genetic interaction between Scn8a and potassium channel genes Kcna1 and Kcnq2.

Voltage-gated sodium and potassium channels regulate the initiation and termination of neuronal action potentials. Gain-of-function mutations of sodium channel Scn8a and loss-of-function mutations of potassium channels Kcna1 and Kcnq2 increase neuronal activity and lead to seizure disorders. We tested the hypothesis that reducing the expression of Scn8a would compensate for loss-of-function mutations of Kcna1 or Kcnq2. Scn8a expression was reduced by the administration of an antisense oligonucleotide (ASO). This treatment lengthened the survival of the Kcn1a and Kcnq2 mutants, and reduced the seizure frequency in the Kcnq2 mutant mice. These observations suggest that reduction of SCN8A may be therapeutic for genetic epilepsies resulting from mutations in these potassium channel genes.

Clinical and Functional Study of a De Novo Variant in the PVP Motif of Kv1.1 Channel Associated with Epilepsy, Developmental Delay and Ataxia.

Mutations in the KCNA1 gene, encoding the voltage-gated potassium channel Kv1.1, have been associated with a spectrum of neurological phenotypes, including episodic ataxia type 1 and developmental and epileptic encephalopathy. We have recently identified a de novo variant in KCNA1 in the highly conserved Pro-Val-Pro motif within the pore of the Kv1.1 channel in a girl affected by early onset epilepsy, ataxia and developmental delay. Other mutations causing severe epilepsy are located in Kv1.1 pore domain. The patient was initially treated with a combination of antiepileptic drugs with limited benefit. Finally, seizures and ataxia control were achieved with lacosamide and acetazolamide. The aim of this study was to functionally characterize Kv1.1 mutant channel to provide a genotype-phenotype correlation and discuss therapeutic options for KCNA1-related epilepsy. To this aim, we transfected HEK 293 cells with Kv1.1 or P403A cDNAs and recorded potassium currents through whole-cell patch-clamp. P403A channels showed smaller potassium currents, voltage-dependent activation shifted by +30 mV towards positive potentials and slower kinetics of activation compared with Kv1.1 wild-type. Heteromeric Kv1.1+P403A channels, resembling the condition of the heterozygous patient, confirmed a loss-of-function biophysical phenotype. Overall, the functional characterization of P403A channels correlates with the clinical symptoms of the patient and supports the observation that mutations associated with severe epileptic phenotype cluster in a highly conserved stretch of residues in Kv1.1 pore domain. This study also strengthens the beneficial effect of acetazolamide and sodium channel blockers in KCNA1 channelopathies.

Kv1.1 preserves the neural stem cell pool and facilitates neuron maturation during adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897­914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.

An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.

Ion Channel Modeling beyond State of the Art: A Comparison with a System Theory-Based Model of the Shaker-Related Voltage-Gated Potassium Channel Kv1.1.

The mathematical modeling of ion channel kinetics is an important tool for studying the electrophysiological mechanisms of the nerves, heart, or cancer, from a single cell to an organ. Common approaches use either a Hodgkin-Huxley (HH) or a hidden Markov model (HMM) description, depending on the level of detail of the functionality and structural changes of the underlying channel gating, and taking into account the computational effort for model simulations. Here, we introduce for the first time a novel system theory-based approach for ion channel modeling based on the concept of transfer function characterization, without a priori knowledge of the biological system, using patch clamp measurements. Using the shaker-related voltage-gated potassium channel Kv1.1 (KCNA1) as an example, we compare the established approaches, HH and HMM, with the system theory-based concept in terms of model accuracy, computational effort, the degree of electrophysiological interpretability, and methodological limitations. This highly data-driven modeling concept offers a new opportunity for the phenomenological kinetic modeling of ion channels, exhibiting exceptional accuracy and computational efficiency compared to the conventional methods. The method has a high potential to further improve the quality and computational performance of complex cell and organ model simulations, and could provide a valuable new tool in the field of next-generation in silico electrophysiology.

Distinct epilepsy phenotypes and response to drugs in KCNA1 gain- and loss-of function variants.

A wide phenotypic spectrum of neurological diseases is associated with KCNA1 (Kv1.1) variants. To investigate the molecular basis of such a heterogeneous clinical presentation and identify the possible correlation with in vitro phenotypes, we compared the functional consequences of three heterozygous de novo variants (p.P403S, p.P405L, and p.P405S) in Kv1.1 pore region found in four patients with severe developmental and epileptic encephalopathy (DEE), with those of a de novo variant in the voltage sensor (p.A261T) identified in two patients with mild, carbamazepine-responsive, focal epilepsy. Patch-clamp electrophysiology was used to investigate the functional properties of mutant Kv1.1 subunits, both expressed as homomers and heteromers with wild-type Kv1.1 subunits. KCNA1 pore mutations markedly decreased (p. P405S) or fully suppressed (p. P403S, p. P405L) Kv1.1-mediated currents, exerting loss-of-function (LoF) effects. By contrast, channels carrying the p.A261T variant exhibited a hyperpolarizing shift of the activation process, consistent with a gain-of-function (GoF) effect. The present results unveil a novel correlation between in vitro phenotype (GoF vs LoF) and clinical course (mild vs severe) in KCNA1-related phenotypes. The excellent clinical response to carbamazepine observed in the patients carrying the A261T variant suggests an exquisite sensitivity of KCNA1 GoF to sodium channel inhibition that should be further explored.