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Generation of human induced pluripotent stem cells (iPSCs) through reprogramming was a transformational change in the field of regenerative medicine that led to new possibilities for drug discovery and cell replacement therapy. Several protocols have been established to differentiate hiPSCs into neuronal lineages. However, low differentiation efficiency is one of the major drawbacks of these approaches. Here, we compared the efficiency of two methods of neuronal differentiation from iPSCs cultured in two different culture media, StemFlex Medium (SFM) and Essential 8 Medium (E8M). The results indicated that iPSCs cultured in E8M efficiently generated different types of neurons in a shorter time and without the growth of undifferentiated nonneuronal cells in the culture as compared with those generated from iPSCs in SFM. Furthermore, these neurons were validated as functional units immunocytochemically by confirming the expression of mature neuronal markers (i.e., NeuN, β tubulin, and Synapsin I) and whole cell patch-clamp recordings. Long-read single-cell RNA sequencing confirms the presence of upper and deep layer cortical layer excitatory and inhibitory neuronal subtypes in addition to small populations of GABAergic neurons in day 30 neuronal cultures. Pathway analysis indicated that our protocol triggers the signaling transcriptional networks important for the process of neuronal differentiation in vivo . NEW & NOTEWORTHY Low differentiation efficiency is one of the major drawbacks of the existing protocols to differentiate iPSCs into neuronal lineages. Here, we present time-efficient and robust approach of neuronal differentiation leading to the generation of functional brain units, cortical layer neurons. We found iPSCs cultured in Essential 8 media (E8M) resulted in neuronal differentiation without the signs of growth of spontaneously differentiated cells in culture at any point in 35 days compared with Stemflex media (SFM).

Loss-of-function mutations in the KCNA1 (Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies ( Sh 5 ). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG ( ether-à-go-go-related gene ) K + channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl, swell ) mediated by the volume-regulated anion channel (VRAC), and the regulatory volume decrease, MLC1's proper function is still unknown. By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca 2+ /Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca 2+ release, potentiates VRAC-mediated ICl, swell . Overall, these findings reveal that MLC1 is a Ca 2+ -regulated protein, linking volume regulation to Ca 2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.

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.

Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2 , E236K, in a Serbian proband with non-progressive congenital ataxia and early onset epilepsy, treated with sodium valproate. To ascertain the pathogenicity of E236K mutation and to verify its sensitivity to 4-AP, we transfected HEK 293 cells with Kv1.2 WT or E236K cDNAs and recorded potassium currents through the whole-cell patch-clamp. In silico analysis supported the electrophysiological data. E236K channels showed voltage-dependent activation shifted towards negative potentials and slower kinetics of deactivation and activation compared with Kv1.2 WT. Heteromeric Kv1.2 WT+E236K channels, resembling the condition of the heterozygous patient, confirmed a mixed gain- and loss-of-function (GoF/LoF) biophysical phenotype. 4-AP inhibited both Kv1.2 and E236K channels with similar potency. Homology modeling studies of mutant channels suggested a reduced interaction between the residue K236 in the S2 segment and the gating charges at S4. Overall, the biophysical phenotype of E236K channels correlates with the mild end of the clinical spectrum reported in patients with GoF/LoF defects. The response to 4-AP corroborates existing evidence that KCNA2 -disorders could benefit from variant-tailored therapeutic approaches, based on functional studies.

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18 , and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K + permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H + (pHi) and extracellular K + concentration [K + ] o , and hence provides a link between pHi and [K + ] o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K + ] o -dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.

The KCNA1 gene encodes the α subunit of the voltage-gated Kv1.1 potassium channel that critically regulates neuronal excitability in the central and peripheral nervous systems. Mutations in KCNA1 have been classically associated with episodic ataxia type 1 (EA1), a movement disorder triggered by physical and emotional stress. Additional features variably reported in recent years include epilepsy, myokymia, migraine, paroxysmal dyskinesia, hyperthermia, hypomagnesemia, and cataplexy. Interestingly, a few individuals with neuromyotonia, either isolated or associated with skeletal deformities, have been reported carrying variants in the S2-S3 transmembrane segments of Kv1.1 channels in the absence of any other symptoms. Here, we have identified by whole-exome sequencing a novel de novo variant, T268K, in KCNA1 in a boy displaying recurrent episodes of neuromyotonia, muscle hypertrophy, and skeletal deformities. Through functional analysis in heterologous cells and structural modeling, we show that the mutation, located at the extracellular end of the S3 helix, causes deleterious effects, disrupting Kv1.1 function by altering the voltage dependence of activation and kinetics of deactivation, likely due to abnormal interactions with the voltage sensor in the S4 segment. Our study supports previous evidence suggesting that specific residues within the S2 and S3 segments of Kv1.1 result in a distinctive phenotype with predominant musculoskeletal presentation.

Inherited and sporadic mutations in genes encoding for brain ion channels, affecting membrane expression or biophysical properties, have been associated with neurodevelopmental disorders characterized by epilepsy, cognitive and behavioral deficits with significant phenotypic and genetic heterogeneity. Over the years, the screening of a growing number of patients and the functional characterization of newly identified mutations in ion channels genes allowed to recognize new phenotypes and to widen the clinical spectrum of known diseases. Furthermore, advancements in understanding disease pathogenesis at atomic level or using patient-derived iPSCs and animal models have been pivotal to orient therapeutic intervention and to put the basis for the development of novel pharmacological options for drug-resistant disorders. In this review we will discuss major improvements and critical issues concerning neurodevelopmental disorders caused by dysfunctions in brain sodium, potassium, calcium, chloride and ligand-gated ion channels., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mutations in the KCNK18 gene that encodes the TRESK K2P potassium channel have previously been linked with typical familial migraine with aura. Recently, an atypical clinical case has been reported in which a male individual carrying the p.Trp101Arg (W101R) missense mutation in the KCNK18 gene was diagnosed with intellectual disability and migraine with brainstem aura. Here we report the functional characterization of this new missense variant. This mutation is located in a highly conserved residue close to the selectivity filter, and our results show although these mutant channels retain their K + selectivity and calcineurin-dependent regulation, the variant causes an overall dramatic loss of TRESK channel function as well as an initial dominant-negative effect when co-expressed with wild-type channels in Xenopus laevis oocytes. The dramatic functional consequences of this mutation thereby support a potentially pathogenic role for this variant and provide further insight into the relationship between the structure and function of this ion channel.

Investigating the Shaker-related K + channel Kv1.1, the dysfunction of which is responsible for episodic ataxia 1 (EA1), at the functional and molecular level provides valuable understandings on normal channel dynamics, structural correlates underlying voltage-gating, and disease-causing mechanisms. Most studies focused on apparently functional amino acid residues composing voltage-gated K + channels, neglecting the simplest ones. Glycine at position 311 of Kv1.1 is highly conserved both evolutionarily and within the Kv channel superfamily, is located in a region functionally relevant (the S4-S5 linker), and results in overt disease when mutated (p.G311D). By mutating the G311 residue to aspartate, we show here that the channel voltage-gating, activation, deactivation, inactivation, and window currents are markedly affected. In silico, modeling shows this glycine residue is strategically placed at one end of the linker helix which must be free to both bend and move past other portions of the protein during the channel's opening and closing. This is befitting of a glycine residue as its small neutral side chain allows for movement unhindered by interaction with any other amino acid. Results presented reveal the crucial importance of a distinct glycine residue, within the S4-S5 linker, in the voltage-dependent electromechanical coupling that control channel gating.

Episodic ataxia type 2 (EA2) is characterized by paroxysmal attacks of ataxia with typical onset in childhood or early adolescence. The disease is associated with mutations in the voltage-gated calcium channel alpha 1A subunit (Cav2.1) that is encoded by the CACNA1A gene. However, previously unrecognized atypical symptoms and the genetic overlap existing between EA2, spinocerebellar ataxia type 6, familial hemiplegic migraine type 1, and other neurological diseases blur the genotype/phenotype correlations, making a differential diagnosis difficult to formulate correctly and delaying early therapeutic intervention. Here we report a new clinical phenotype of a CACNA1A -associated disease characterized by absence epilepsy occurring during childhood. However, much later in life the patient displayed non-episodic, slowly progressive gait ataxia. Gene panel sequencing for hereditary ataxias led to the identification of a novel heterozygous CACNA1A mutation (c.1913 + 2T > G), altering the donor splice site of intron 14. This genetic defect was predicted to result in an in-frame deletion removing 44 amino acids from the voltage-gated calcium channel Cav2.1. An RT-PCR analysis of cDNA derived from patient skin fibroblasts confirmed the skipping of the entire exon 14. Furthermore, two-electrode voltage-clamp recordings performed from Xenopus laevis oocytes expressing a wild-type versus mutant channel showed that the genetic defect caused a complete loss of channel function. This represents the first description of distinct clinical manifestations that remarkably expand the genetic and phenotypic spectrum of CACNA1A- related diseases and should be considered for an early diagnosis and effective therapeutic intervention.

Kv1.1 belongs to the Shaker subfamily of voltage-gated potassium channels and acts as a critical regulator of neuronal excitability in the central and peripheral nervous systems. KCNA1 is the only gene that has been associated with episodic ataxia type 1 (EA1), an autosomal dominant disorder characterized by ataxia and myokymia and for which different and variable phenotypes have now been reported. The iterative characterization of channel defects at the molecular, network, and organismal levels contributed to elucidating the functional consequences of KCNA1 mutations and to demonstrate that ataxic attacks and neuromyotonia result from cerebellum and motor nerve alterations. Dysfunctions of the Kv1.1 channel have been also associated with epilepsy and kcna1 knock-out mouse is considered a model of sudden unexpected death in epilepsy. The tissue-specific association of Kv1.1 with other Kv1 members, auxiliary and interacting subunits amplifies Kv1.1 physiological roles and expands the pathogenesis of Kv1.1-associated diseases. In line with the current knowledge, Kv1.1 has been proposed as a novel and promising target for the treatment of brain disorders characterized by hyperexcitability, in the attempt to overcome limited response and side effects of available therapies. This review recounts past and current studies clarifying the roles of Kv1.1 in and beyond the nervous system and its contribution to EA1 and seizure susceptibility as well as its wide pharmacological potential.

Glioblastoma multiforme (GBM) is the most common and malignant of the glial tumors. The world-wide estimates of new cases and deaths annually are remarkable, making GBM a crucial public health issue. Despite the combination of radical surgery, radio and chemotherapy prognosis is extremely poor (median survival is approximately 1 year). Thus, current therapeutic interventions are highly unsatisfactory. For many years, GBM-induced brain oedema and inflammation have been widely treated with dexamethasone (DEX), a synthetic glucocorticoid (GC). A number of studies have reported that DEX also inhibits GBM cell proliferation and migration. Nevertheless, recent controversial results provided by different laboratories have challenged the widely accepted dogma concerning DEX therapy for GBM. Here, we have reviewed the main clinical features and genetic and epigenetic abnormalities underlying GBM. Finally, we analyzed current notions and concerns related to DEX effects on cerebral oedema, cancer cell proliferation and migration and clinical outcome.

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K + channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na + -activated K + channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K + currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease. NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI., (Copyright © 2017 the American Physiological Society.)

Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3-S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10mV toward positive potentials and kinetics of activation slowed by ~2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K + channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel's loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Background: Voltage-gated potassium channels are highly diverse proteins representing the most complex class of voltage-gated ion channels from structural and functional perspectives. Deficiency of these channels usually results in various human disorders., Objectives: To describe a novel autosomal recessive syndrome associated with KCNA4 deficiency leading to congenital cataract, abnormal striatum, intellectual disability and attention deficit hyperactivity disorder., Methods: We used SNP arrays, linkage analyses, autozygosity mapping, whole-exome sequencing, RT-PCR and two-electrode voltage-clamp recording., Results: We identified a missense variant (p.Arg89Gln) in KCNA4 in four patients from a consanguineous family manifesting a novel syndrome of congenital cataract, abnormal striatum, intellectual disability and attention deficit hyperactivity disorder. The variant was fully segregated with the disease and absent in 747 ethnically matched exomes. Xenopus oocytes were injected with human Kv1.4 wild-type mRNA, R89Q and WT/R89Q channels. The wild type had mean current amplitude that was significantly greater than those recorded from the cells expressing the same amount of mutant mRNA. Co-expression of the wild type and mutant mRNAs resulted in mean current amplitude that was significantly different from that of the wild type. RT-PCR indicated that KCNA4 is present in mouse brain, lens and retina. KCNA4 interacts with several molecules including synaptotagmin I, DLG1 and DLG2. The channel co-localises with cholinergic amacrine and rod bipolar cells in rats and is widely distributed in the central nervous system. Based on previous studies, the channel is highly expressed in outer retina, rod inner segments, hippocampus and concentrated in axonal membranes., Conclusion: KCNA4 (Kv1.4) is implicated in a novel syndrome characterised by striatal thinning, congenital cataract and attention deficit hyperactivity disorder. Our study highlights potassium channels' role in ocular and neuronal genetics., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy".

Episodic ataxia type 1 (EA1) is a K(+) channelopathy characterized by a broad spectrum of symptoms. Generally, patients may experience constant myokymia and dramatic episodes of spastic contractions of the skeletal muscles of the head, arms, and legs with loss of both motor coordination and balance. During attacks additional symptoms may be reported such as vertigo, blurred vision, diplopia, nausea, headache, diaphoresis, clumsiness, stiffening of the body, dysarthric speech, and difficulty in breathing. These episodes may be precipitated by anxiety, emotional stress, fatigue, startle response or sudden postural changes. Epilepsy is overrepresented in EA1. The disease is inherited in an autosomal dominant manner, and genetic analysis of several families has led to the discovery of a number of point mutations in the voltage-dependent K(+) channel gene KCNA1 (Kv1.1), on chromosome 12p13. To date KCNA1 is the only gene known to be associated with EA1. Functional studies have shown that these mutations impair Kv1.1 channel function with variable effects on channel assembly, trafficking and biophysics. Despite the solid evidence obtained on the molecular mechanisms underlying EA1, how these cause dysfunctions within the central and peripheral nervous systems circuitries remains elusive. This review summarizes the main breakthrough findings in EA1, discusses the neurophysiological mechanisms underlying the disease, current therapies, future challenges and opens a window onto the role of Kv1.1 channels in central nervous system (CNS) and peripheral nervous system (PNS) functions.

Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, and is notable for spreading so effectively through the brain parenchyma to make complete surgical resection virtually impossible, and prospect of life dismal. Several ion channels have been involved in GBM migration and invasion, due to their critical role in supporting volume changes and Ca(2+) influx occuring during the process. The large-conductance, Ca(2+)-activated K (BK) channels, markedly overexpressed in biopsies of patients with GBMs and in GBM cell lines, have attracted much interest and have been suggested to play a central role in cell migration and invasion as candidate channels for providing the ion efflux and consequent water extrusion that allow cell shrinkage during migration. Available experimental data on the role of BK channel in migration and invasion are not consistent though. While BK channels block typically resulted in inhibition of cell migration or in no effect, their activation would either enhance or inhibit the process. This short review reexamines the relevant available data on the topic, and presents a unifying paradigm capable of reconciling present discrepancies. According to this paradigm, BK channels would not contribute to migration under conditions where the [Ca(2+)] i is too low for their activation. They will instead positively contribute to migration for intermediate [Ca(2+)] i , insufficient as such to activate BK channels, but capable of predisposing them to cyclic activation following oscillatory [Ca(2+)] i increases. Finally, steadily active BK channels because of prolonged high [Ca(2+)] i would inhibit migration as their steady activity would be unsuitable to match the cyclic cell volume changes needed for proper cell migration.

Trigeminal ganglion (TG) neurons are functionally and morphologically heterogeneous, and the molecular basis of this heterogeneity is still not fully understood. Here we describe experiments showing that a subpopulation of neurons expresses a delayed-rectifying K(+) current (IDRK) with a characteristically high (nanomolar) sensitivity to the dihydroquinoline CP339,818 (CP). Although submicromolar CP has previously been shown to selectively block Kv1.3 and Kv1.4 channels, the CP-sensitive IDRK found in TG neurons could not be associated with either of these two K(+) channels. It could neither be associated with Kv2.1 channels homomeric or heteromerically associated with the Kv9.2, Kv9.3, or Kv6.4 subunits, whose block by CP, tested using two-electrode voltage-clamp recordings from Xenopus oocytes, resulted in the low micromolar range, nor to the Kv7 subfamily, given the lack of blocking efficacy of 3 μM XE991. Within the group of multiple-firing neurons considered in this study, the CP-sensitive IDRK was preferentially expressed in a subpopulation showing several nociceptive markers, such as small membrane capacitance, sensitivity to capsaicin, and slow afterhyperpolarization (AHP); in these neurons the CP-sensitive IDRK controls the membrane resting potential, the firing frequency, and the AHP duration. A biophysical study of the CP-sensitive IDRK indicated the presence of two kinetically distinct components: a fast deactivating component having a relatively depolarized steady-state inactivation (IDRKf) and a slow deactivating component with a more hyperpolarized V1/2 for steady-state inactivation (IDRKs)., (Copyright © 2015 the American Physiological Society.)

Autism spectrum disorders (ASDs) are characterized by impaired ability to properly implement environmental stimuli that are essential to achieve a state of social and cultural exchange. Indeed, the main features of ASD are impairments of interpersonal relationships, verbal and non-verbal communication and restricted and repetitive behaviors. These aspects are often accompanied by several comorbidities such as motor delay, praxis impairment, gait abnormalities, insomnia, and above all epilepsy. Genetic analyses of autistic individuals uncovered deleterious mutations in several K(+) channel types strengthening the notion that their intrinsic dysfunction may play a central etiologic role in ASD. However, indirect implication of K(+) channels in ASD has been also reported. For instance, loss of fragile X mental retardation protein (FMRP) results in K(+) channels deregulation, network dysfunction and ASD-like cognitive and behavioral symptoms. This review provides an update on direct and indirect implications of K(+) channels in ASDs. Owing to a mounting body of evidence associating a channelopathy pathogenesis to autism and showing that nearly 500 ion channel proteins are encoded by the human genome, we propose to classify ASDs - whose susceptibility is significantly enhanced by ion channels defects, either in a monogenic or multigenic condition - in a new category named " c hannel A utism S pectrum D isorder" (channelASD; cASD) and introduce a new taxonomy (e.g., Kv x.y-channelASD and likewise Nav x.y-channelASD, Cav x.y-channelASD; etc.). This review also highlights some degree of clinical and genetic overlap between K(+) channelASDs and K(+) channelepsies, whereby such correlation suggests that a subcategory characterized by a channelASD-channelepsy phenotype may be distinguished. Ultimately, this overview aims to further understand the different clinical subgroups and help parse out the distinct biological basis of autism that are essential to establish patient-tailored treatments.

Episodic ataxia type 1 (EA1) is an autosomal dominant K(+) channelopathy which manifests with short attacks of cerebellar ataxia and dysarthria, and may also show interictal myokymia. Episodes can be triggered by emotional or physical stress, startle response, sudden postural change or fever. Here we describe a 31-year-old man displaying markedly atypical symptoms, including long-lasting attacks of jerking muscle contractions associated with hyperthermia, severe migraine, and a relatively short-sleep phenotype. A single nucleotide change in KCNA1 (c.555C>G) was identified that changes a highly conserved residue (p.C185W) in the first transmembrane segment of the voltage-gated K(+) channel Kv1.1. The patient is heterozygous and the mutation was inherited from his asymptomatic mother. Next generation sequencing revealed no variations in the CACNA1A, CACNB4, KCNC3, KCNJ10, PRRT2 or SCN8A genes of either the patient or mother, except for a benign variant in SLC1A3. Functional analysis of the p.C185W mutation in KCNA1 demonstrated a deleterious dominant-negative phenotype where the remaining current displayed slower activation kinetics, subtle changes in voltage-dependence and faster recovery from slow inactivation. Structural modeling also predicts the C185W mutation to be functionally deleterious. This description of novel clinical features, associated with a Kv1.1 mutation highlights a possibly unrecognized relationship between K(+) channel dysfunction, hyperthermia and migraine in EA1, and suggests that thorough assessments for these symptoms should be carefully considered for all patients affected by EA1.

The malignancy of glioblastoma multiform (GBM), the most common and aggressive form of human brain tumors, strongly correlates with the presence of hypoxic areas, but the mechanisms controlling the hypoxia-induced aggressiveness are still unclear. GBM cells express a number of ion channels whose activity supports cell volume changes and increases in the cytosolic Ca(2+) concentration, ultimately leading to cell proliferation, migration or death. In several cell types it has previously been shown that low oxygen levels regulate the expression and activity of these channels, and more recent data indicate that this also occurs in GBM cells. Based on these findings, it may be hypothesized that the modulation of ion channel activity or expression by the hypoxic environment may participate in the acquisition of the aggressive phenotype observed in GBM cells residing in a hypoxic environment. If this hypothesis will be confirmed, the use of available ion channels modulators may be considered for implementing novel therapeutic strategies against these tumors.

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism-epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channel's surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin-proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management., (© The Author 2014. Published by Oxford University Press.)

Background: Most biological tissues are characterized by high morphological and functional cell heterogeneity. To investigate this heterogeneity at the molecular level, scientists have tried to associate specific sets of molecular markers (molecular profiles) to functionally distinct cell subpopulations, evaluating their expression using immunochemistry and in situ hybridization techniques., New Method: We propose here a novel analysis that allows the estimation of the frequency of cells expressing distinct molecular profiles starting from data on the co-expression of two markers at a time. In order to facilitate the application of the proposed analysis, we developed and make available a user-friendly window-based software., Results: We successfully applied the analytical method to experimental data from adult rat sensory neurons. In a first application we subgrouped DRG neurons in 11 subpopulations on the basis of the co-expression of 6 molecular markers (the TRPs type V1, A1, and M8 and the trks type A, B, and C). In a second application we found that while rat DRG have significant frequencies of peptidergic/IB4-negative and non-peptidergic/IB4-positive nociceptors, rat TG neurons lack almost completely these two subpopulations., Comparison With Existing Methods: The analytical method here proposed overcomes the limitations of the presently available experimental techniques, most of which can assess the co-expression of only few molecular markers at a time., Conclusions: This new method will allow a better understanding of the molecular and cellular heterogeneity of tissues in normal and pathological conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The activity of voltage-gated K(+) channels (Kv) can be dynamically modulated by several events, including neurotransmitter stimulated biochemical cascades mediated by G protein-coupled receptors such as 5-HT2 receptors (5-HT2Rs). Activation of 5-HT2A/CR inhibits the Shaker-like K(+) channels Kv1.1 and Kv1.2, and this modulation involves the dual coordination of both RPTPα and distinct tyrosine kinases coupled to this receptor; 5-HT2Rs-mediated modulation of Kv channels controls glutamate release onto prefrontal cortex neurons that might play critical roles in neurophysiological, neurological, and psychiatric conditions. Noticeably, hallucinogens modulate Kv channel activity, acting at 5-HT2R. Hence, comprehensive knowledge of 5-HT2R signaling through modulation of distinct K(+) channels is a pivotal step in the direction that will enable scientists to discover novel 5-HT functions and dysfunctions in the brain and to identify original therapeutic targets.

K(+) channels are important determinants of seizure susceptibility. These membrane proteins, encoded by more than 70 genes, make the largest group of ion channels that fine-tune the electrical activity of neuronal and non-neuronal cells in the brain. Their ubiquity and extremely high genetic and functional diversity, unmatched by any other ion channel type, place K(+) channels as primary targets of genetic variations or perturbations in K(+)-dependent homeostasis, even in the absence of a primary channel defect. It is therefore not surprising that numerous inherited or acquired K(+) channels dysfunctions have been associated with several neurologic syndromes, including epilepsy, which often generate confusion in the classification of the associated diseases. Therefore, we propose to name the K(+) channels defects underlying distinct epilepsies as "K(+) channelepsies," and introduce a new nomenclature (e.g., Kx.y-channelepsy), following the widely used K(+) channel classification, which could be also adopted to easily identify other channelopathies involving Na(+) (e.g., Nav x.y-phenotype), Ca(2+) (e.g., Cav x.y-phenotype), and Cl(-) channels. Furthermore, we discuss novel genetic defects in K(+) channels and associated proteins that underlie distinct epileptic phenotypes in humans, and analyze critically the recent progress in the neurobiology of this disease that has also been provided by investigations on valuable animal models of epilepsy. The abundant and varied lines of evidence discussed here strongly foster assessments for variations in genes encoding for K(+) channels and associated proteins in patients with idiopathic epilepsy, provide new avenues for future investigations, and highlight these proteins as critical pharmacological targets.

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K(+) channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1(V408A/+)). Here, we investigated the neuromuscular transmission of Kv1.1(V408A/+) ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve-muscle from Kv1.1(+/+) and Kv1.1(V408A/+) mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca(2+) signals that occurred abnormally only in preparations dissected from Kv1.1(V408A/+) mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca(2+) homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K(+) channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aim: Atopic dermatitis (AD) is a common, chronic relapsing inflammatory skin disease characterized by dry skin and variable pruritus sometimes associated with allergic disease in other organs as asthma and rhinoconjunctivitis. AD affects deeply the Quality of Life, thus can be extremely disabling and may cause psychological problems for both affected children and their families., Methods: In order to investigate the estimated prevalence of the disease and the beliefs of the Italian pediatricians, a group of 437 Italian family pediatricians covering a population of almost 380000 children participated in a study based on a questionnaire of 38 items., Results: According to answers of the participants, the incidence of AD has been estimated around 10% of the population and food allergy is believed to be the trigger of the acute phase of the disease in infants. As a second opinion, dermatologists are consulted more frequently than allergologists., Conclusion: The use of emollients is advised in general whilst topical corticosteroids treatment is prescribed only in selected cases; more than 50% of pediatricians do not prescribe topical calcineurin inhibitors.

The inwardly-rectifying potassium channel Kir4.1 is a major player in the astrocyte-mediated regulation of [K(+)](o) in the brain, which is essential for normal neuronal activity and synaptic functioning. KCNJ10, encoding Kir4.1, has been recently linked to seizure susceptibility in humans and mice, and is a possible candidate gene for Autism Spectrum Disorders (ASD). In this study, we performed a mutational screening of KCNJ10 in 52 patients with epilepsy of "unknown cause" associated with impairment of either cognitive or communicative abilities, or both. Among them, 14 patients fitted the diagnostic criteria for ASD. We identified two heterozygous KCNJ10 mutations (p.R18Q and p.V84M) in three children (two unrelated families) with seizures, ASD, and intellectual disability. The mutations replaced amino acid residues that are highly conserved throughout evolution and were undetected in about 500 healthy chromosomes. The effects of mutations on channel activity were functionally assayed using a heterologous expression system. These studies indicated that the molecular mechanism contributing to the disorder relates to an increase in either surface-expression or conductance of the Kir4.1 channel. Unlike previous syndromic associations of genetic variants in KCNJ10, the pure neuropsychiatric phenotype in our patients suggests that the new mutations affect K(+) homeostasis mainly in the brain, by acting through gain-of-function defects. Dysfunction in astrocytic-dependent K(+) buffering may contribute to autism/epilepsy phenotype, by altering neuronal excitability and synaptic function, and may represent a new target for novel therapeutic approaches., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 subunits.

The molecular identity of ion channels which confer PCO(2)/pH sensitivity in the brain is unclear. Heteromeric Kir4.1/Kir5.1 channels are highly sensitive to inhibition by intracellular pH and are widely expressed in several brainstem nuclei involved in cardiorespiratory control, including the locus coeruleus. This has therefore led to a proposed role for these channels in neuronal CO(2) chemosensitivity. To examine this, we generated mutant mice lacking the Kir5.1 (Kcnj16) gene. We show that although locus coeruleus neurons from Kcnj16((+/+)) mice rapidly respond to cytoplasmic alkalinization and acidification, those from Kcnj16((-/-)) mice display a dramatically reduced and delayed response. These results identify Kir5.1 as an important determinant of PCO(2)/pH sensitivity in locus coeruleus neurons and suggest that Kir5.1 may be involved in the response to hypercapnic acidosis.

Shaker-like (KV1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (KV2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to KV1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of KV channel gating by contributing, at least in part, to the relative flexibility of this motif.

Episodic ataxia type 1 (EA1) is a rare human neurological syndrome characterized by continuous myokymia and attacks of generalized ataxia that can be triggered by abrupt movements, emotional stress and fatigue. An Italian family has been identified where related members displayed continuous myokymia, episodes of ataxia, attacks characterized by myokymia only, and neuromyotonia. A novel missense mutation (F414C), in the C-terminal region of the K(+) channel Kv1.1, was identified in the affected individuals. The mutant homotetrameric channels were non-functional in Xenopus laevis oocytes. In addition, heteromeric channels resulting from the co-expression of wild-type Kv1.1 and Kv1.1(F414C), or wild-type Kv1.2 and Kv1.1(F414C) subunits displayed reduced current amplitudes and altered gating properties. This indicates that the pathogenic effect of this KCNA1 mutation is likely to be related to the defective functional properties we have identified.

Episodic ataxia type 1 (EA1) is a Shaker-like channelopathy characterized by continuous myokymia and attacks of imbalance with jerking movements of the head, arms, and legs. Although altered expression and gating properties of Kv1.1 channels underlie EA1, several disease-causing mechanisms remain poorly understood. It is likely that Kv1.1, Kv1.4, and Kvbeta1.1 subunits form heteromeric channels at hippocampal mossy fiber boutons from which Zn(2+) ions are released into the synaptic cleft in a Ca(2+)-dependent fashion. The sensitivity of this macromolecular channel complex to Zn(2+) is unknown. Here, we show that this heteromeric channel possesses a high-affinity (<10 muM) and a low-affinity (<0.5 mM) site for Zn(2+), which are likely to regulate channel availability at distinct presynaptic membranes. Furthermore, the EA1 mutation F184C, located within the S1 segment of the Kv1.1 subunit, markedly decreased the equilibrium dissociation constants for Zn(2+) binding to the high- and low-affinity sites. The functional characterization of the Zn(2+) effects on heteromeric channels harboring the F184C mutation also showed that this ion significantly 1) slowed the activation rate of the channel, 2) increased the time to reach peak current amplitude, 3) decreased the rate and amount of current undergoing N-type inactivation, and 4) slowed the repriming of the channel compared with wild-type channels. These results demonstrate that the EA1 mutation F184C will not only sensitize the homomeric Kv1.1 channel to extracellular Zn(2+), but it will also endow heteromeric channels with a higher sensitivity to this metal ion. During the vesicular release of Zn(2+), its effects will be in addition to the intrinsic gating defects caused by the mutation, which is likely to exacerbate the symptoms by impairing the integration and transmission of signals within specific brain areas.

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker-like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kvbeta1.x subunits. Here we show that the human Kvbeta1.1 and Kvbeta1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kvbeta1.1 and Kvbeta1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements.

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.

Voltage-gated potassium channels (Kv) play important roles in neurotransmission, nerve cell excitability and disease. Several missense mutations in the Kv1.1 gene have been associated with episodic ataxia type-1 syndrome (EA-1), which is characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles. In this study we show that I177N, an EA-1 mutation located in S1 segment, alters the expression and gating properties of the channel expressed in Xenopus oocytes. In particular, it reduces ~17-fold the current amplitude, accelerates ~4-fold the deactivation kinetics of the channel and shifts the voltage dependence of activation ~60 mV to more depolarized potentials. Single-channel recordings also showed that the mean open duration of I177N channels was ~2.6-fold smaller than the wild-type. These results demonstrate that both reduced current levels and specific gating defects are the likely causes of EA-1 symptoms in patients bearing the I177N mutation. Furthermore, the data suggest that the I177N substitution may alter the gating properties of the channel that are specifically defined by the S1 segment.