Kir7.1 disease mutant T153I within the inner pore affects K+ conduction.

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid side-chain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full-length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wild-type channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel's narrow inner pore in regulating conductance.

Dissecting activation steps in P2X7 receptors.

P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.

Charged Residues in the Membrane Anchor of the Pestiviral Erns Protein Are Important for Processing and Secretion of Erns and Recovery of Infectious Viruses.

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.

Neurofibromatosis 1 - Mutant microglia exhibit sexually-dimorphic cyclic AMP-dependent purinergic defects.

As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/-) mice, purinergic control of phagocytosis was only affected in male Nf1+/- mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/-, microglia. These defects resulted from Nf1+/- male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/- microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.

Encoding Membrane-Potential-Based Memory within a Microbial Community.

Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be encoded in simpler organisms like bacteria. Here, we show that single-cell-level memory patterns can be imprinted in bacterial biofilms by light-induced changes in the membrane potential. We demonstrate that transient optical perturbations generate a persistent and robust potassium-channel-mediated change in the membrane potential of bacteria within the biofilm. The light-exposed cells respond in an anti-phase manner, relative to unexposed cells, to both natural and induced oscillations in extracellular ion concentrations. This anti-phase response, which persists for hours following the transient optical stimulus, enables a direct single-cell resolution visualization of spatial memory patterns within the biofilm. The ability to encode robust and persistent membrane-potential-based memory patterns could enable computations within prokaryotic communities and suggests a parallel between neurons and bacteria.

In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

The HV 1 voltage-gated proton (HV 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of HV 1 is Zn2+ . Externally applied ZnCl2 drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn2+ and Cd2+ on HV 1 from Nicoletia phytophila, NpHV 1. We introduced mutations at potential Zn2+ coordination sites and measured Zn2+ inhibition in different extracellular pH, with Zn2+ concentrations up to 1000 µm. Zn2+ inhibition in NpHV 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn2+ inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn2+ in the S3-S4 linker. A simple Hodgkin Huxley model of NpHV 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpHV 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.

Voltage-dependent activation of Rac1 by Nav 1.5 channels promotes cell migration.

Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.

17ß-Estradiol Increases Arcuate KNDy Neuronal Sensitivity to Ghrelin Inhibition of the M-Current in Female Mice.

Obesity and anorexia result in dysregulation of the hypothalamic-pituitary-gonadal axis, negatively impacting reproduction. Ghrelin, secreted from the stomach, potentially mediates negative energy states and neuroendocrine control of reproduction by acting through the growth hormone secretagogue receptor (GHSR). GHSR is expressed in hypothalamic arcuate (ARC) Kisspeptin/Neurokinin B (Tac2)/Dynorphin (KNDy) neurons. Ghrelin is known to inhibit the M-current produced by KCNQ channels in other ARC neurons. In addition, we have shown 17ß-estradiol (E2) increases Ghsr expression in KNDy neurons 6-fold and increases the M-current in NPY neurons. We hypothesize that E2 increases GHSR expression in KNDy neurons to increase ghrelin sensitivity during negative energy states. Furthermore, we suspect ghrelin targets the M-current in KNDy neurons to control reproduction and energy homeostasis. We utilized ovariectomized Tac2-EGFP adult female mice, pretreated with estradiol benzoate (EB) or oil vehicle and performed whole-cell-patch-clamp recordings to elicit the M-current in KNDy neurons using standard activation protocols in voltage-clamp. Using the selective KCNQ channel blocker XE-991 (40 µM) to target the M-current, oil- and EB-treated mice showed a decrease in the maximum peak current by 75.7 ± 13.8 pA (n = 10) and 68.0 ± 14.7 pA (n = 11), respectively. To determine the actions of ghrelin on the M-current, ghrelin was perfused (100 nM) in oil- and EB-treated mice resulting in the suppression of the maximum peak current by 58.5 ± 15.8 pA (n = 9) and 59.2 ± 11.9 pA (n = 9), respectively. KNDy neurons appeared more sensitive to ghrelin when pretreated with EB, revealing that ARC KNDy neurons are more sensitive to ghrelin during states of high E2.

Arabidopsis H+-ATPase AHA1 controls slow wave potential duration and wound-response jasmonate pathway activation.

Electrogenic proton pumps have been implicated in the generation of slow wave potentials (SWPs), damage-induced membrane depolarizations that activate the jasmonate (JA) defense pathway in leaves distal to wounds. However, no defined H+-ATPases have been shown to modulate these electrical signals. Pilot experiments revealed that the proton pump activator fusicoccin attenuated SWP duration in Arabidopsis Using mutant analyses, we identified Arabidopsis H+-ATPase 1 (AHA1) as a SWP regulator. The duration of the repolarization phase was strongly extended in reduced function aha1 mutants. Moreover, the duration of SWP repolarization was shortened in the presence of a gain-of-function AHA1 allele. We employed aphid electrodes to probe the effects of the aha1 mutation on wound-stimulated electrical activity in the phloem. Relative to the wild type, the aha1-7 mutant increased the duration and reduced the amplitudes of electrical signals in sieve tube cells. In addition to affecting electrical signaling, expression of the JA pathway marker gene JAZ10 in leaves distal to wounds was enhanced in aha1-7 Consistent with this, levels of wound-response jasmonoyl-isoleucine were enhanced in the mutant, as was defense against a lepidopteran herbivore. The work identifies a discrete member of the P-type ATPase superfamily with a role in leaf-to-leaf electrical signaling and plant defense.

Inhibition of miR-337-3p involved in the protection of CoCl2 -induced injury in PC12 cells via activating JAK2/STAT3 signaling pathway.

OBJECTIVE: To investigate the possibility of microRNA (miR)-337-3p in the protection of hypoxia-induced injury in PC12 cells via modulating the JAK2/STAT3 signaling pathway. METHODS: Dual-luciferase reporter assay analyzed the relationship between the miR-337-3p and JAK2. PC12 cells were divided into normal, CoCl2 , CoCl2 + NC, CoCl2 + inhibitors, CoCl2 + JAK2, and CoCl2 + mimics + JAK2 groups. Then, PC12 cell viability and apoptosis were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Annexin-V-fluorescein isothiocyanate/propidium iodide methods. Quantitative real-time polymerase chain reaction and Western blot analysis were used to determine expressions. Besides, the intracellular reactive oxygen species (ROS) was examined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) while the mitochondrial membrane potential (MMP) by using JC-1. RESULTS: The negative targeting relationship between miR-337-3p and JAK2 was confirmed. When compared with the normal group, miR-337-3p was increased while JAK2 and STAT3 were decreased in CoCl2 -induced PC12 cells, with decreased cell viability. Moreover, either miR-337-3p inhibitor or JAK2 overexpression could partially reverse CoCl2 -induced decrease in PC12 cell viability. Besides, CoCl2 could also trigger PC12 cell apoptosis by increasing cleaved caspase 3 and Bax but decreasing Bcl-2 and Bcl-XL, which, however, were abolished with the transfection of miR-337-3p inhibitors or lentivirus transfection to activate JAK2. Compared with the CoCl2 group, the average of fluorescent signals of ROS in the CoCl2 + inhibitors group and the CoCl2 + JAK2 group was lower, while the activities of superoxide dismutase, catalase, glutathione peroxidase, and total anti-oxidative capacity were higher, together with an increase in MMP. CONCLUSION: Inhibiting miR-337-3p could activate the JAK2/STAT3 signaling pathway to suppress CoCl 2 -induced cytotoxicity and apoptosis and ameliorate oxidative stress and MMP in PC12 cells.

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials.

The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2 -interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.

The Neuronal Ischemic Tolerance Is Conditioned by the Tp53 Arg72Pro Polymorphism.

Cerebral preconditioning (PC) confers endogenous brain protection after stroke. Ischemic stroke patients with a prior transient ischemic attack (TIA) may potentially be in a preconditioned state. Although PC has been associated with the activation of pro-survival signals, the mechanism by which preconditioning confers neuroprotection is not yet fully clarified. Recently, we have described that PC-mediated neuroprotection against ischemic insult is promoted by p53 destabilization, which is mediated by its main regulator MDM2. Moreover, we have previously described that the human Tp53 Arg72Pro single nucleotide polymorphism (SNP) controls susceptibility to ischemia-induced neuronal apoptosis and governs the functional outcome of patients after stroke. Here, we studied the contribution of the human Tp53 Arg72Pro SNP on PC-induced neuroprotection after ischemia. Our results showed that cortical neurons expressing the Pro72-p53 variant exhibited higher PC-mediated neuroprotection as compared with Arg72-p53 neurons. PC prevented ischemia-induced nuclear and cytosolic p53 stabilization in Pro72-p53 neurons. However, PC failed to prevent mitochondrial p53 stabilization, which occurs in Arg72-p53 neurons after ischemia. Furthermore, PC promoted neuroprotection against ischemia by controlling the p53/active caspase-3 pathway in Pro72-p53, but not in Arg72-p53 neurons. Finally, we found that good prognosis associated to TIA within 1 month prior to ischemic stroke was restricted to patients harboring the Pro72 allele. Our findings demonstrate that the Tp53 Arg72Pro SNP controls PC-promoted neuroprotection against a subsequent ischemic insult by modulating mitochondrial p53 stabilization and then modulates TIA-induced ischemic tolerance.

Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte-derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα-driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

Identification and characterization of two zebrafish Twik related potassium channels, Kcnk2a and Kcnk2b.

KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies.

Maladaptive cortical hyperactivity upon recovery from experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) patients exhibit neuropsychological symptoms in early disease despite the immune attack occurring predominantly in white matter and spinal cord. It is unclear why neurodegeneration may start early in the disease and is prominent in later stages. We assessed cortical microcircuit activity by employing spiking-specific two-photon Ca2+ imaging in proteolipid protein-immunized relapsing-remitting SJL/J mice in vivo. We identified the emergence of hyperactive cortical neurons in remission only, independent of direct immune-mediated damage and paralleled by elevated anxiety. High levels of neuronal activity were accompanied by increased caspase-3 expression. Cortical TNFα expression was mainly increased by excitatory neurons in remission; blockade with intraventricular infliximab restored AMPA spontaneous excitatory postsynaptic current frequencies, completely recovered normal neuronal network activity patterns and alleviated elevated anxiety. This suggests a dysregulation of cortical networks attempting to achieve functional compensation by synaptic plasticity mechanisms, indicating a link between immune attack and early start of neurodegeneration.

Directional coupling of oligodendrocyte connexin-47 and astrocyte connexin-43 gap junctions.

Intercellular communication via gap junction channels between oligodendrocytes and between astrocytes as well as between these cell types is essential to maintain the integrity of myelin in the central nervous system. Oligodendrocyte gap junction connexin-47 (Cx47) is a key element in this crosstalk and indeed, mutations in human Cx47 cause severe myelin disorders. However, the permeation properties of channels of Cx47 alone and in heterotypic combination with astrocyte Cx43 remain unclear. We show here that Cx47 contains three extra residues at 5' amino-terminus that play a critical role in the channel pore structure and account for relative low ionic conductivity, cationic permselectivity and voltage-gating properties of oligodendrocyte-oligodendrocyte Cx47 channels. Regarding oligodendrocyte-astrocyte coupling, heterotypic channels formed by Cx47 with Cx43 exhibit ionic and chemical rectification, which creates a directional diffusion barrier for the movement of ions and larger negatively charged molecules from cells expressing Cx47 to those with Cx43. The restrictive permeability of Cx47 channels and the diffusion barrier of Cx47-Cx43 channels was abolished by a mutation associated with leukodystrophy, the Cx47P90S, suggesting a novel pathogenic mechanism underlying myelin disorders that involves alterations in the panglial permeation.

Attenuating M-current suppression in vivo by a mutant Kcnq2 gene knock-in reduces seizure burden and prevents status epilepticus-induced neuronal death and epileptogenesis.

OBJECTIVES: The M-current is a low-threshold voltage-gated potassium current generated by Kv7 subunits that regulates neural excitation. It is important to note that M-current suppression, induced by activation of Gq-coupled neurotransmitter receptors, can dynamically regulate the threshold of action-potential firing and firing frequency. Here we sought to directly examine whether M-current suppression is involved in seizures and epileptogenesis. METHODS: Kv7.2 knock-in mice lacking the key protein kinase C (PKC) phosphorylation acceptor site for M-current suppression were generated by introducing an alanine substitution at serine residue 559 of mouse Kv7.2, mKv7.2(S559A). Basic electrophysiologic properties of the M-current between wild-type and Kv7.2(S559A) knock-in mice were analyzed in primary cultured neurons. Homozygous Kv7.2(S559A) knock-in mice were used to evaluate the protective effect of mutant Kv7.2 channel against chemoconvulsant-induced seizures. In addition, pilocarpine-induced neuronal damage and spontaneously recurrent seizures were evaluated after equivalent chemoconvulsant-induced status epilepticus was achieved by coadministration of the M-current-specific channel inhibitor, XE991. RESULT: Neurons from Kv7.2(S559A) knock-in mice showed normal basal M-currents. Knock-in mice displayed reduced M-current suppression when challenged by a muscarinic agonist, oxotremorine-M. Kv7.2(S559A) mice were resistant to chemoconvulsant-induced seizures with no mortality. Administration of XE991 transiently exacerbated seizures in knock-in mice equivalent to those of wild-type mice. Valproate, which disrupts neurotransmitter-induced M-current suppression, showed no additional anticonvulsant effect in Kv7.2(S559A) mice. After experiencing status epilepticus, Kv7.2(S559A) knock-in mice did not show seizure-induced cell death or spontaneous recurring seizures. SIGNIFICANCE: This study provides evidence that neurotransmitter-induced suppression of M-current generated by Kv7.2-containing channels exacerbates behavioral seizures. In addition, prompt recovery of M-current after status epilepticus prevents subsequent neuronal death and the development of spontaneously recurrent seizures. Therefore, prompt restoration of M-current activity may have a therapeutic benefit for epilepsy.

SCN4A as modifier gene in patients with myotonic dystrophy type 2.

A patient with an early severe myotonia diagnosed for Myotonic Dystrophy type 2 (DM2) was found bearing the combined effects of DM2 mutation and Nav1.4 S906T substitution. To investigate the mechanism underlying his atypical phenotype,whole-cell patch-clamp in voltage- and current-clamp mode was performed in myoblasts and myotubes obtained from his muscle biopsy. Results characterizing the properties of the sodium current and of the action potentials have been compared to those obtained in muscle cells derived from his mother, also affected by DM2, but without the S906T polymorphism. A faster inactivation kinetics and a +5 mV shift in the availability curve were found in the sodium current recorded in patient's myoblasts compared to his mother. 27% of his myotubes displayed spontaneous activity. Patient's myotubes showing a stable resting membrane potential had a lower rheobase current respect to the mother's while the overshoot and the maximum slope of the depolarizing phase of action potential were higher. These findings suggest that SCN4A polymorphisms may be responsible for a higher excitability of DM2 patients sarcolemma, supporting the severe myotonic phenotype observed. We suggest SCN4A as a modifier factor and that its screening should be performed in DM2 patients with uncommon clinical features.