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Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K+ channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca2+). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies.

Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in KCNQ2 and KCNQ3, two genes encoding for potassium channel subunits. A large family with nine members affected by BFNC is described in the present study. All affected members of this family carry a novel deletion/insertion mutation in the KCNQ2 gene (c.761_770del10insA), which determines a premature truncation of the protein. In addition, in the family of the proposita's father, a novel sequence variant (c.2687A>G) in KCNQ3 leading to the p.N821S amino acid change was detected. When heterologously expressed in Chinese hamster ovary cells, KCNQ2 subunits carrying the mutation failed to form functional potassium channels in homomeric configuration and did not affect channels formed by KCNQ2 and/or KCNQ3 subunits. On the other hand, homomeric and heteromeric potassium channels formed by KCNQ3 subunits carrying the p.N821S variant were indistinguishable from those formed by wild-type KCNQ3 subunits. Finally, the current density of the cells mimicking the double heterozygotic condition for both KCNQ2 and KCNQ3 alleles of the proband was decreased by approximately 25% when compared to cells expressing only wild-type alleles. Collectively, these results suggest that, in the family investigated, the KCNQ2 mutation is responsible for the BFNC phenotype, possibly because of haplo-insufficiency, whereas the KCNQ3 variant is functionally silent, a result compatible with its lack of segregation with the BFNC phenotype.

Increased activity of plasma membrane K+ channels, leading to decreased cytoplasmic K+ concentrations, occurs during neuronal cell death. In the present study, we showed that the neurotoxic beta-amyloid peptide Abeta(25-35) caused a dose-dependent (0.1-10 microm) and time-dependent (> 12 h) enhancement of both inactivating and non-inactivating components of voltage-dependent K+ (VGK) currents in nerve growth factor-differentiated rat phaeochromocytoma (PC-12) cells and primary rat hippocampal neurones. Similar effects were exerted by Abeta(1-42), but not by the non-neurotoxic Abeta(35-25) peptide. Abeta(25-35) and Abeta(1-42) caused an early (15-20 min) increase in intracellular Ca(2+) concentration. This led to an increased production of reactive oxygen species (ROS), which peaked at 3 h and lasted for 24 h; ROS production seemed to trigger the VGK current increase as vitamin E (50 microm) blocked both the Abeta(25-35)- and Abeta(1-42)-induced ROS increase and VGK current enhancement. Inhibition of protein synthesis (cycloheximide, 1 microg/mL) and transcription (actinomycin D, 50 ng/mL) blocked Abeta(25-35)-induced VGK current enhancement, suggesting that this potentiation is mediated by transcriptional activation induced by ROS. Interestingly, the specific nuclear factor-kappaB inhibitor SN-50 (5 microm), but not its inactive analogue SN-50M (5 microm), fully counteracted Abeta(1-42)- or Abeta(25-35)-induced enhancement of VGK currents, providing evidence for a role of this family of transcription factors in regulating neuronal K+ channel function during exposure to Abeta.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. The SLC7A7 gene, mutated in LPI, encodes the y(+)LAT-1 protein, which is the light subunit of the heterodimeric CAA transporter in which 4F2hc is the heavy chain subunit. Co-expression of 4F2hc and y(+)LAT-1 induces the y(+)L activity. This activity is also exerted by another complex composed of 4F2hc and y(+)LAT-2, the latter encoded by the SLC7A6 gene and more ubiquitously expressed than SLC7A7. On the basis of both the pattern of expression and the transport activity, y(+)LAT-2 might compensate for CAA transport when y(+)LAT-1 is defective. By expression in Xenopus laevis oocytes and mammalian cells, we functionally analysed two SLC7A7 mutants, E36del and F152L, respectively, the former displaying a partial dominant-negative effect. The results of the present study provide further insight into the molecular pathogenesis of LPI: a putative multiheteromeric structure of both [4F2hc/y(+)LAT-1] and [4F2hc/y(+)LAT-2], and the interference between y(+)LAT-1 and y(+)LAT-2 proteins. This interference can explain why the compensatory mechanism, that is, an increased expression of SLC7A6 as seen in lymphoblasts from LPI patients, may not be sufficient to restore the y(+)L system activity.

Summary The present article reports on the recent findings addressing the molecular basis for some rare but serious cardiovascular side-effects exerted by some non-sedating H1-blocking antihistamines. These latest developments, which have allowed fundamental insights into the role played by a specific class of potassium channels, the so-called human ether-a-gogo-related gene (HERG) channels, in the regulation of cardiac action potential duration and rhythm regulation, have also opened new areas of investigation into its participation in neuronal and endocrine (adenohypophyseal, chromaffin, and pancreatic) cells functioning.

This review addresses novel concepts of histamine H1-receptor function and attempts to relate them to the anti-inflammatory effects of H1-antihistamines. Furthermore, the molecular mechanisms underlying the cardiotoxic effects of H1-antihistamines are discussed. H1-receptors are G-protein-coupled-receptors (GPCRs), the inactive and active conformations of which coexist in equilibrium. The degree receptor activation in the absence of histamine is its 'constitutive activity'. In this two-state model, histamine acts as an agonist by combining with and stabilizing the activated conformation of the H1-receptor to shift the equilibrium towards the activated state. Drugs classified previously as antagonists act as either inverse agonists or neutral antagonists. Inverse agonists combine with and stabilize the inactive conformation of the receptor to shift the equilibrium towards the inactive state. Thus, they may down-regulate constitutive receptor activity, even in the absence of histamine. Neutral antagonists combine equally with both conformations of the receptor, do not affect basal receptor activity but do interfere with agonist binding. All H1-antihistamines examined to date are inverse agonists. As the term 'H1-receptor antagonists' is obviously erroneous, we suggest that it be replaced by 'H1-antihistamines'. The observations that H1-receptors modulate NF-kappaB activation and that there are complex interactions between GPCRs, has allowed us to postulate receptor dependent-mechanisms for some anti-inflammatory effects of H1-antihistamines, e.g. inhibition of ICAM-1 expression and the effects of bradykinin. Finally, the finding that blockade of HERG1 K+ channels is the mechanism by which some H1-antihistamines may cause cardiac arrhythmias has allowed the development of preclinical tests to predict such activity.

The K(+) channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K(+) channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopus oocytes. Substitution of the S(5)-S(6) linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S(5)-S(6) linker generated K(+) channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K(+) currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S(5)-S(6) linker region of hERG1 K(+) channels are crucial players in ROS-induced modulation of hERG1 K(+) channels.

Large conductance, calcium-activated potassium channels [big potassium (BK) channel] consist of a tetramer of pore-forming α-subunit and distinct accessory β-subunits (β1-4) that modify the channel's properties. In this study, we analyzed the effects of BK channel activators and blockers on glutamate and γ-aminobutyric acid (GABA) release from synaptosomes isolated from the cerebral cortices or trigeminal caudal nuclei (TCN) of rats. Real-time polymerase chain reaction was used to characterize BK channel α and β(1-4) subunit expression in the cortex and in the trigeminal ganglia (TG), whose neurons project primary terminal afferents into the TCN. Immunocytochemistry was used to localize these subunits on cortical and TCN synaptosomes. The BK channels regulating [(3)H]D-aspartate release from primary afferent nerve terminals projecting into the TCN displayed limited sensitivity to iberiotoxin, whereas those expressed on cortical synaptosomes were highly sensitive to this toxin. BK channels did not appear to be present on GABAergic nerve terminals from the TCN since [(3)H]-γ-aminobutyric acid release in this model was unaffected by BK channel activators or blockers. Gene expression studies revealed expression levels of the α subunit in the TG that were only 31.2 ± 2.1% of those found in cortical tissues. The β4 subunit was the accessory subunit expressed most abundantly in both the cortex and TG. Levels of β1 and β2 were low in both these areas although β2 expression in the TG was higher than that found in the cortex. Immunocytochemistry experiments showed that co-localization of α and β4 subunits (the accessory subunit most abundantly expressed in both brain areas) was more common in TCN synaptosomes than in cortical synaptosomes. On the basis of these findings, it is reasonable to hypothesize that BK channels expressed on glutamatergic terminals in the TCN and cortex have distinct pharmacological profiles, which probably reflect different α and β subunit combinations. Channels in the cortex seem to be composed mainly of α subunits and to a lesser degree by α and β4 subunits, whereas in the TG the α + β4 combination seems to prevail (although α and/or α + β2 channels cannot be excluded). In light of the BK channels' selective control of excitatory transmission and their pharmacological diversity, their effects on primary glutamatergic afferents projecting to TCN represent a potential target for drug therapy of migraines and other types of orofacial pain.

In the present study, the neuroprotective effects of the adipokine leptin, and the molecular mechanism involved, have been studied in rat and mice cortical neurons exposed to N-methyl- d -aspartate (NMDA) in vitro . In rat cortical neurons, leptin elicited neuroprotective effects against NMDA-induced cell death, which were concentration-dependent (10–100 ng/ml) and largest when the adipokine was preincubated for 2 h before the neurotoxic stimulus. In both rat and mouse cortical neurons, leptin-induced neuroprotection was fully antagonized by paxilline (Pax, 0.01–1 μM) and iberiotoxin (Ibtx, 1–100 nM), with EC 50 s of 38 ± 10 nM and 5 ± 2 nM for Pax and Ibtx, respectively, close to those reported for Pax- and Ibtx-induced Ca 2+ - and voltage-activated K + channels (Slo1 BK channels) blockade; the BK channel opener NS1619 (1–30 μM) induced a concentration-dependent protection against NMDA-induced excitotoxicity. Moreover, cortical neurons from mice lacking one or both alleles coding for Slo1 BK channel pore-forming subunits were insensitive to leptin-induced neuroprotection. Finally, leptin exposure dose-dependently (10–100 ng/ml) increased intracellular Ca 2+ levels in rat cortical neurons. In conclusion, our results suggest that Slo1 BK channel activation following increases in intracellular Ca 2+ levels is a critical step for leptin-induced neuroprotection in NMDA-exposed cortical neurons in vitro , thus highlighting leptin-based intervention via BK channel activation as a potential strategy to counteract neurodegenerative diseases.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) are decreased both during myotube formation in vitro from murine C2C12 myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-2-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, 2-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP2), underlies the effects of 2-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP2 levels in C2C12 cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that 2-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

When compared to older first-generation antihistamines, second-generation antihistamines are characterized by improved selectivity for histamine receptors, absence of sedation, and, possibly, antiallergic properties distinct from their antihistaminic activity. Such a pharmacologic profile, arising from specific pharmacodynamic and pharmacokinetic properties, bears an obvious clinical advantage for the therapy of allergic diseases; thus second-generation antihistamines have become the treatment of choice for chronic urticaria and allergic rhinitis. Despite such a therapeutic advantage, adverse cardiovascular effects associated with the use of some congeners belonging to the class of second-generation antihistamines (particularly terfenadine and astemizole) have been reported, and a major concern about the therapeutic selection of antihistamines now is represented by their potentially severe arrhythmogenic properties. This article reviews the recent advances in the understanding of the pathogenesis and etiology of the cardiotoxic actions of some second-generation antihistamines, underlining their molecular actions at the level of K+ channels controlling the cardiac action potential. In particular, emphasis is given to the interaction of second-generation antihistamines with the K+ channels encoded by the human ether-a-gogo-related gene (HERG), which is crucially involved in the repolarization process of the cardiac action potential. This review will also focus on the recent concerns over the potential cardiac adverse effects of first-generation H1 receptor blockers. The present exploration of the molecular basis of the adverse cardiac effects of antihistamines shows how important contributions in deciphering such complex phenomenon have emerged from disciplines and techniques not traditionally related to immunology, such as molecular genetics and cellular electrophysiology; it seems possible to anticipate that the exchange of results from such different disciplines in the future will provide patients and physicians with medications with improved therapeutic efficacy and safety for the clinical management of allergic diseases.

Ventricular arrhythmias are rare but life-threatening side effects of therapy with the second-generation H1 receptor antagonists terfenadine and astemizole. Blockade of the K+ channels encoded by the Human Ether-a-go-go-Related Gene 1 (HERG1) K+ channels, which is the molecular basis of the cardiac repolarizing current IKr, by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. In the present study, the potential blocking ability of the novel second-generation H1 receptor antagonist mizolastine of the HERG1 K+ channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH-SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. Mizolastine blocked HERG1 K+ channels expressed in Xenopus oocytes with an estimated IC50 of 3.4 μM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K+ channel were 72.4±11.9 s for 3 μM mizolastine, and 1361±306 s for 1 μM astemizole. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose-related inhibitory action on IHERG1, with an IC50 of 350±76 nM. Furthermore, mizolastine dose-dependently inhibited HERG1 K+ channels constitutively expressed in SH-SY5Y human neuroblastoma clonal cells. The results of the present study suggest that the novel second-generation H1 receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K+ channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1-inhibitory action. British Journal of Pharmacology (2000) 131, 1081–1088; doi:10.1038/sj.bjp.0703654

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (KIR), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs+), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured andin situhippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood–brain barrier. cDNA amplification showed that cortical astrocytes selectively expressHERG1, but notHERG2–3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Despite the enormous success of second generation antihistamines, in the mid-1980s, about 10 years after their introduction in the market, several reports appeared in the literature indicating the rare occurrence of a form of polymorphic ventricular dysrhythmia, the 'torsade de pointes', after the administration of astemizole or terfenadine. This cardiac side-effect has been interpreted as a consequence of the interference of these drugs with cardiac K+ channels involved in action potential repolarization, and in particular with the IKr component of the cardiac repolarizing current. As the K+ channels encoded by the human ether-a-gogo-related gene (HERG) seem to represent the molecular basis of IKr, this cardiac K+ channel was soon recognized as a primary target for second generation antihistamine-induced proarrhythmic effects. In fact, both terfenadine and astemizole have been shown to block HERG K+ channels in a concentration range similar to that found in the plasma of subjects with cardiotoxic manifestations. However, no correlation can be found between the ability to prolong the cardiac action potential duration and the H1-antagonistic activity by several antihistamines, suggesting that HERG blockade and cardiotoxic potential are not class properties of second generation antihistamines. In fact, other molecules such as cetirizine, loratadine, acrivastine, and fexofenadine seem to lack both cardiotoxic potential and HERG-blocking ability at therapeutically relevant concentrations. The marked heterogeneity displayed by second generation antihistamines in their ability to prolong the cardiac action potential duration and to block HERG K+ channels might be of considerable therapeutical significance for those patients at risk of developing cardiac dysrhythmias and in need of therapy with H1-receptor blockers; it also emphasizes the importance of an evaluation of the possible blockade of HERG K+ channels during the early developmental phases of novel compounds belonging to this therapeutical class.

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKβ₂₋₆₄, a novel variant of AaTXKβ₁₋₆₄, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKβ₂₋₆₄, but not AaTXKβ₁₋₆₄, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKβ₂₋₆₄ also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKβ₂₋₆₄-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.

Mutations in the K V 7.2 gene encoding for voltage-dependent K + channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S 4 domain of K V 7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K V 7.2 and/or K V 7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were ( i ) more pronounced for channels incorporating R213Q- than R213W-carrying K V 7.2 subunits; ( ii ) proportional to the number of mutant subunits incorporated; and ( iii ) fully restored by the neuronal K v 7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K + channel impairment, and set the preclinical basis for the potential use of K v 7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K V 7.2 encephalopathy.

Summary Purpose To dissect the genetics of benign familial epilepsies of the first year of life and to assess the extent of the genetic overlap between benign familial neonatal seizures (BFNS), benign familial neonatal-infantile seizures (BFNIS), and benign familial infantile seizures (BFIS). Methods Families with at least two first-degree relatives affected by focal seizures starting within the first year of life and normal development before seizure onset were included. Families were classified as BFNS when all family members experienced neonatal seizures, BFNIS when the onset of seizures in family members was between 1 and 4 months of age or showed both neonatal and infantile seizures, and BFIS when the onset of seizures was after 4 months of age in all family members. SCN2A, KCNQ2, KCNQ3, PPRT2 point mutations were analyzed by direct sequencing of amplified genomic DNA. Genomic deletions involving KCNQ2 and KCNQ3 were analyzed by multiple-dependent probe amplification method. Key Findings A total of 46 families including 165 affected members were collected. Eight families were classified as BFNS, 9 as BFNIS, and 29 as BFIS. Genetic analysis led to the identification of 41 mutations, 14 affecting KCNQ2, 1 affecting KCNQ3, 5 affecting SCN2A, and 21 affecting PRRT2. The detection rate of mutations in the entire cohort was 89%. In BFNS, mutations specifically involve KCNQ2. In BFNIS two genes are involved (KCNQ2, six families; SCN2A, two families). BFIS families are the most genetically heterogeneous, with all four genes involved, although about 70% of them carry a PRRT2 mutation. Significance Our data highlight the important role of KCNQ2 in the entire spectrum of disorders, although progressively decreasing as the age of onset advances. The occurrence of afebrile seizures during follow-up is associated with KCNQ2 mutations and may represent a predictive factor. In addition, we showed that KCNQ3 mutations might be also involved in families with infantile seizures. Taken together our data indicate an important role of K-channel genes beyond the typical neonatal epilepsies. The identification of a novel SCN2A mutation in a family with infantile seizures with onset between 6 and 8 months provides further confirmation that this gene is not specifically associated with BFNIS and is also involved in families with a delayed age of onset. Our data indicate that PRRT2 mutations are clustered in families with BFIS. Paroxysmal kinesigenic dyskinesia emerges as a distinctive feature of PRRT2 families, although uncommon in our series. We showed that the age of onset of seizures is significantly correlated with underlying genetics, as about 90% of the typical BFNS families are linked to KCNQ2 compared to only 3% of the BFIS families, for which PRRT2 represents the major gene.

The use of -blockers (BB) in heart failure (HF) has been considered a contradiction for many years. Considering HF simply as a state of inadequate systolic function, BB were contraindicated because of their negative effects on myocardial contractility. Nevertheless, evidence collected in the past years have suggested that additional mechanisms, such as compensatory neuro-humoral hyperactivation or inflammation, could participate in the pathogenesis of this complex disease. Indeed, chronic activation of the sympathetic nervous system, although initially compensating the reduced cardiac output from the failing heart, increases myocardial oxygen demand, ischemia and oxidative stress; moreover, high catecholamine levels induce peripheral vasoconstriction and increase both cardiac pre- and after-load, thus determining additional stress to the cardiac muscle (1). As a consequence of such a different view of the pathogenic mechanisms of HF, the efficacy of BB in the treatment of HF has been investigated in numerous clinical trials. Results from these trials highlighted BB as valid therapeutic tools in HF, providing rational basis for their inclusion in many HF treatment guidelines. However, controversy still exists about their use, in particular with regards to the selection of specific molecules, since BB differ in terms of adrenergic -receptors selectivity, adjunctive effects on -receptors, and effects on reactive oxygen species and inflammatory cytokines production. Further concerns about the heterogeneity in the response to , as well as the use in specific patients, are matter of debate among clinicians. In this review, we will recapitulate the pharmacological properties and the classification of BB, and the alteration of the adrenergic system occurring during HF that provide a rationale for their use; we will also focus on the possible molecular mechanisms, such as genetic polymorphisms, underlying the different efficacy of molecules belonging to this class.

Voltage-dependent type 7 K + (K V 7 or KCNQ) channels modulate the excitability of neurons and muscle cells. The aims of the present study were to investigate the motor effects of K V 7 channel modulators and the expression of K V 7 channels in the human taenia coli . The effects of K V 7 channel modulators on the muscle tone of human taenia coli strips were investigated under nonadrenergic non-nitrergic conditions by organ bath studies. Gene expression and tissue localisation of channels were studied by real-time PCR and immunohistochemistry, respectively. Under basal conditions, the K V 7 channel blocker XE-991 induced concentration-dependent contractions, with mean EC 50 and E max of 18.7 μM and 30.5% respectively of the maximal bethanechol-induced contraction, respectively. The K V 7 channel activators retigabine and flupirtine concentration-dependently relaxed the taenia coli , with mean EC 50 s of 19.2 μM and 29.9 μM, respectively. Retigabine also relaxed bethanechol-precontracted strips, with maximal relaxations of 79.2% of the bethanecol-induced precontraction. The motor effects induced by the K V 7 channel modulators were not affected by tetrodotoxin or ω-conotoxin GVIA. XE-991 greatly reduced retigabine- and flupirtine-induced relaxations. Transcripts encoded by all KCNQ genes were detected in the taenia coli , with KCNQ4 showing the highest expression levels. K V 7.4 channels were clearly visualised by immunohistochemistry in colonic epithelium, circular muscle layer and taenia coli . K V 7 channels appear to contribute to the resting muscle tone of the human taenia coli . In addition, K V 7 channel activators significantly relax the taenia coli . Thus, they could be useful therapeutic relaxant agents for colonic motor disorders.

Kv7.4-potassium channel expression plays a permissive role in skeletal muscle differentiation. The transcriptional repressor REST controls the changes in Kv7.4 levels during myogenesis by binding to regulatory regions in the Kv7.4 gene. This mechanism may be a target for intervention against abnormal repair and differentiation of skeletal muscle., Changes in the expression of potassium (K+) channels is a pivotal event during skeletal muscle differentiation. In mouse C2C12 cells, similarly to human skeletal muscle cells, myotube formation increased the expression of Kv7.1, Kv7.3, and Kv7.4, the last showing the highest degree of regulation. In C2C12 cells, Kv7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In Kv7.4-silenced cells, the differentiation-promoting effect of the Kv7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for Kv7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5′ untranslated region and in the first intron of the Kv7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that Kv7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K+ channel subunit.

The novel antiepileptic and neuroprotective drug felbamate (1 mM) caused a marked inhibition of voltage-dependent Na+ currents expressed in Xenopus oocytes upon injection of the cRNA encoding alpha-subunits from rat and human brain. This inhibition was present only if felbamate was perfused on the intracellular side of the membrane. In addition, felbamate seems to preferentially bind to and stabilize the inactivated state of the channel, resembling the action of local anesthetics. This study provides an additional mechanism by which felbamate might exert its wide-spectrum anticonvulsant and neuroprotective action.

Abstract Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as I Kr , the rapidly activating component of the repolarizing outward cardiac K + current, I K . I Kr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K + (K v ) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K + current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K + rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC 50 =533 nmol/L at 40 mV and 20°C). The drug has no significant blocking effect on the human cardiac K v channels hK v 1.2, hK v 1.4, hK v 1.5, or hK v 2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau=1.17 seconds at −80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.

Objective: To advance the understanding of KCNQ2 encephalopathy genotype–phenotype relationships and to begin to assess the potential of selective KCNQ channel openers as targeted treatments. Methods: We retrospectively studied 23 patients with KCNQ2 encephalopathy, including 11 treated with ezogabine (EZO). We analyzed the genotype–phenotype relationships in these and 70 previously described patients. Results: The mean seizure onset age was 1.8 ± 1.6 (SD) days. Of the 20 EEGs obtained within a week of birth, 11 showed burst suppression. When new seizure types appeared in infancy (15 patients), the most common were epileptic spasms (n = 8). At last follow-up, seizures persisted in 9 patients. Development was delayed in all, severely in 14. The KCNQ2 variants identified introduced amino acid missense changes or, in one instance, a single residue deletion. They were clustered in 4 protein subdomains predicted to poison tetrameric channel functions. EZO use (assessed by the treating physicians and parents) was associated with improvement in seizures and/or development in 3 of the 4 treated before 6 months of age, and 2 of the 7 treated later; no serious side effects were observed. Conclusions: KCNQ2 variants cause neonatal-onset epileptic encephalopathy of widely varying severity. Pathogenic variants in epileptic encephalopathy are clustered in “hot spots” known to be critical for channel activity. For variants causing KCNQ2 channel loss of function, EZO appeared well tolerated and potentially beneficial against refractory seizures when started early. Larger, prospective studies are needed to enable better definition of prognostic categories and more robust testing of novel interventions. Classification of evidence: This study provides Class IV evidence that EZO is effective for refractory seizures in patients with epilepsy due to KCNQ2 encephalopathy.

Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti-inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain-inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca²⁺](i)) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA-induced effects was also studied.[Ca²⁺](i) was evaluated by single-cell microfluorimetry in differentiated F11 cells. Activation of TRPV1 channels was assessed by imaging and patch-clamp techniques in CHO cells transiently-transfected with rat TRPV1 cDNA.In F11 cells, PEA (1-30 μM) dose-dependently increased [Ca²⁺](i). The TRPV1 antagonists capsazepine (1 μM) and SB-366791 (1 μM), as well as the PPARα antagonist GW-6471 (10 μM), inhibited PEA-induced [Ca²⁺](i) increase; blockers of cannabinoid receptors were ineffective. PEA activated TRPV1 channels heterologously expressed in CHO cells; this effect appeared to be mediated at least in part by PPARα. When compared with CAP, PEA showed similar potency and lower efficacy, and caused stronger TRPV1 currents desensitization. Sub-effective PEA concentrations, closer to those found in vivo, counteracted CAP- and BK-induced [Ca²⁺](i) transients, as well as CAP-induced TRPV1 activation.Activation of PPARα and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca²⁺](i) transients in sensory neurons. Differential TRPV1 activation and desensitization by CAP and PEA might contribute to their distinct pharmacological profile, possibly translating into potentially relevant clinical differences.

Background Gamma-aminobutyric acid (GABA) and glutamate (GLU) are involved in nociceptive signals processing in the trigeminal system. In this study, we investigated the influence of excitatory transmission on GABA release in nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). Methods We utilize biochemical (superfused synaptosomes loaded with [3H]GABA) and morphological (immunofluorescence experiments with specific antibody) techniques. Results Our results show that GLU potentiates the release of [3H]GABA evoked by 9, 15 and 30 mM [K+]e; 15 mM [K+]e-evoked [3H]GABA release was also reinforced by domoate and kainate (KA), two naturally occurring GLU-receptor agonists. The enhancement of 15 mM [K+]e-evoked [3H]GABA release produced by 100 μM KA was abolished by NBQX, a mixed AMPA/KA receptor antagonist, but was not affected by GYKI52466, a selective AMPA receptor antagonist. ATPA, a selective agonist for KA receptors containing the GLUK1 subunit, had no effect on depolarization-induced [3H]GABA release, and UBP310, which selectively antagonizes these same receptors, failed to reverse the KA-induced potentiation of 15 mM [K+]e-evoked [3H]GABA release. The KA-induced potentiation was also unaffected by concanavalin A (10 μM), a positive allosteric modulator of GLUK1- and GLUK2-containing KA receptors. Immunofluorescence experiments revealed that GABAergic nerve terminals in the TCN differentially expressed GLUK subunits, with GLUK2/3-positive terminals being twice more abundant than GLUK1-containing synaptosomes. Conclusions These findings indicate that pre-synaptic KA receptors facilitating GABA release from TCN nerve terminals mainly express GLUK2/GLUK3 subunits, supporting the notion that different types of KA receptors are involved in the various stages of pain transmission.

The possibility of the fabrication of organic devices suitable to be applied in bio-sensing fields depends largely on the availability of organic compounds displaying robust electrical properties even in aqueous solutions and effective biocompatibility features. In this paper, we report about the good cellular biocompatibility and the electrical response stability in an ionic medium of n-type organic transistors based on the recently developed PDI-8CN(2) oligomer. The biocompatibility has been tested by analyzing the adhesion and viability of two different cell lines, human epithelial HeLa cells and murine neuronal F11 cells, on PDI-8CN(2) films grown by organic molecular beam deposition (OMBD) on SiO2 substrates. The effect of film thickness on cell attachment was also tested. Uncoated SiO2 substrates were used as control surfaces and sexithiophene (T6) as device testing control. Moreover, the possible toxicity of -CN groups of PDI-8CN(2) was tested on HeLa cell cultures, using PDI-8 and T6 molecules as controls. Results showed that, although at high concentration these organic compounds are toxic in solution, if they are presented in form of film, cell lines can attach and grow on them. The electrical response stability of PDI-8CN(2) transistors in a cellular culture medium characterized by high concentrations of ionic species has been also investigated. For this purpose, low-voltage operation devices with V-GS ranging from -5 V to 5 V, able to strongly reduce the influence of Faradaic currents coming from the electrical operation in an highly ionic environment, have been fabricated on 35 nm thick SiO2 layers and electrically characterized. These results are useful to experimentally define the main critical issues to be further addressed for the fabrication of reliable bio-sensors based on organic transistors. (C) 2012 American Institute of Physics

In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine.

Kv7 potassium channels regulate excitability in neuronal, sensory, and muscular cells. Here, we describe their molecular architecture, physiological roles, and involvement in genetically determined channelopathies highlighting their relevance as targets for pharmacological treatment of several human disorders.

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

Objective: To describe a new FHM kindred, and to analyse the functional consequences of the disease-associated ATP1A2 p.G301R mutation in human cellular models grown at 37°C. Patients and methods: Seven patients were clinically evaluated and gave informed consent for molecular analysis. Extra-pyramidal rigidity of the limbs was present in four subjects and in three of them tongue apraxia was also observed. ATP1A2 and CACNA1A were analysed by direct sequencing. Functional consequences of the mutation were investigated by cell viability assays, Western blots, and immunocytochemistry. Three-dimensional models of the human Na+/K+-ATPase α2 subunit were generated by homology modelling using SWISS-MODEL. Findings: Analysis of ATP1A2 showed a heterozygous mutation, c.901G>A predicting the replacement of arginine for glycine at residue 301 (p.G301R). Functional analysis suggested that the mutation completely abolished Na+/K+-ATPase function. Conclusions: The phenotypic spectrum of our FHM2 family includes some peculiar features. Functional data confirm that Na+/K+-ATPase haploinsufficiency caused by the ATP1A2 p.G301R mutation is responsible for FHM in the described family.

Graphical abstract, Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13–25% or reduced by 11–21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.

Despite the availability of over 20 antiepileptic drugs, about 30% of epileptic patients do not achieve seizure control. Thus, identification of additional molecules targeting novel molecular mechanisms is a primary effort in today's antiepileptic drug research. This paper reviews the pharmacological development of retigabine, an antiepileptic drug with a novel mechanism of action, namely the activation of voltage-gated potassium channels of the Kv7 subfamily. These channels, which act as widespread regulators of intrinsic neuronal excitability and of neurotransmitter-induced network excitability changes, are currently viewed among the most promising targets for anticonvulsant pharmacotherapy. In particular, the present work reviews the pathophysiological role of Kv7 channels in neuronal function, the molecular mechanisms involved in the Kv7 channel-opening action of retigabine, the activity of retigabine in preclinical in vitro and in vivo studies predictive of anticonvulsant activities, and the clinical status of development for this drug as an add-on treatment for pharmacoresistant epilepsy. Particular efforts are devoted to highlighting the potential advantages and disadvantages of retigabine when compared with currently available compounds, in order to provide a comprehensive assessment of its role in therapy for treatment-resistant epilepsies.

Changes in the expression of potassium channels regulate skeletal muscle development. The purpose of this study was to investigate the expression profile and pharmacological role of K(v)7 voltage-gated potassium channels in skeletal muscle differentiation, proliferation, and survival after myotoxic insults. Transcripts for all K(v)7 genes (K(v)7.1-K(v)7.5) were detected by polymerase chain reaction (PCR) and/or real-time PCR in murine C(2)C(12) myoblasts; K(v)7.1, K(v)7.3, and K(v)7.4 transcripts were up-regulated after myotube formation. Western blot experiments confirmed K(v)7.2, K(v)7.3, and K(v)7.4 subunit expression, and the up-regulation of K(v)7.3 and K(v)7.4 subunits during in vitro differentiation. In adult skeletal muscles from mice and humans, K(v)7.2 and K(v)7.3 immunoreactivity was mainly localized at the level of intracellular striations positioned between ankyrinG-positive triads, whereas that of K(v)7.4 subunits was largely restricted to the sarcolemmal membrane. In C(2)C(12) cells, retigabine (10 microM), a specific activator of neuronally expressed K(v)7.2 to K(v)7.5 subunits, reduced proliferation, accelerated myogenin expression, and inhibited the myotoxic effect of mevastatin (IC(50) approximately 7 microM); all these effects of retigabine were prevented by the K(v)7 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 muM). These data collectively highlight neural K(v)7 channels as significant pharmacological targets to regulate skeletal muscle proliferation, differentiation, and myotoxic effects of drugs.

Glutamate (GLU) plays a key role in the transmission and modulation of sensory input to the trigeminal caudal nuclei (TCN). In the present study, we investigated the regulation of previously taken-up [3H] d -aspartate ([3H] d -ASP) release from nerve terminals isolated from rat caudal brainstem, in particular from the zone containing the TCN. TCN neurons can be considered integrative relay neurons linking peripheral and central pain mechanisms. Understanding the mechanisms that control the release of GLU in this area could lead to more effective treatment of migraines and other types of pain associated with the trigeminal nerve. In isolated rat caudal brainstem synaptosomes, exposure to AMPA dose-dependently potentiated [K+]e-stimulated release of [3H] d -ASP (maximum increase: 218 ± 13.08%; EC50: 1.60 ± 0.08 μM). This effect was inhibited by selective AMPA-receptor antagonists (competitive [NBQX] and non-competitive [GYKI52466]) but not by the kainate receptor subunit antagonists NS102 and ACET. AMPA-evoked responses were significantly enhanced by preventing AMPA receptor desensitization with cyclothiazide (10 μM). Basal release of [3H] d -ASP was stimulated by millimolar concentrations of ATP (maximum increase: 197.80 ± 11.85%; EC50: 545 ± 3.15 μM) and by the selective P2X7-receptor agonist benzoylbenzoyl-ATP. ATP also potentiated the release of [3H] d -ASP induced by depolarization. Its effect on basal [3H] d -ASP release was inhibited by the selective P2X7-receptor antagonist A-438079 and by the non-selective antagonist PPADS, but it was only partially suppressed by the ionotropic purinergic receptor antagonist TNP-ATP. Our findings demonstrate that glutamatergic nerve terminals in rat caudal brainstem express AMPA receptors that can facilitate [3H] d -ASP during terminal depolarization and P2X7 receptors that can also enhance this release under basal conditions.

The K(V)7 (KCNQ) subfamily of voltage-gated K(+) channels consists of five members (K(V)7.1-K(V)7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (K(V)7.1) and neuronal (K(V)7.2-K(V)7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal K(V)7 channels (K(V)7.2-K(V)7.5) with the gating currents recorded after K(+) conductance blockade from the same channels. Increasing the recording temperature from 18 degrees C to 28 degrees C accelerated activation/deactivation kinetics of the ionic currents in all homomeric K(V)7 channels (activation Q(10)s at 0 mV were 3.8, 4.1, 8.3 and 2.8 for K(V)7.2, K(V)7.3, K(V)7.4 and K(V)7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28 degrees C, ionic currents carried by K(V)7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in K(V)7.4 and K(V)7.5 channels; the size of the ON gating charges at +40 mV was 1.34 +/- 0.34 nC for K(V)7.4, and 0.79 +/- 0.20 nC for K(V)7.5. At 28 degrees C, K(V)7.4 gating currents had the following salient properties: (1) similar time integral of Q(ON) and Q(OFF), indicating no charge immobilization; (2) a left-shift in the V(1/2) of the Q(ON)/V when compared to the G/V (approximately 50 mV in the presence of 2 mM extracellular Ba(2+)); (3) a Q(ON) decay faster than ionic current activation; and (4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in K(V)7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.

1-Ethyl-benzimidazolinone (EBIO) is a compound originally found to stimulate Cl− secretion from epithelial cells. Targets for EBIO are …

Benign Familial Neonatal Seizures (BFNS) is a rare, autosomal-dominant epilepsy of the newborn caused by mutations in K(v)7.2 (KCNQ2) or K(v)7.3 (KCNQ3) genes encoding for neuronal potassium (K(+)) channel subunits. In this study, we describe a sporadic case of BFNS; the affected child carried heterozygous missense mutations in both K(v)7.2 (D212G) and K(v)7.3 (P574S) alleles. Electrophysiological experiments revealed that the K(v)7.2 D212G substitution, neutralizing a unique negatively-charged residue in the voltage sensor of K(v)7.2 subunits, altered channel gating, leading to a marked destabilization of the open state, a result consistent with structural analysis of the K(v)7.2 subunit, suggesting a possible pathogenetic role for BFNS of this K(v)7.2 mutation. By contrast, no significant functional changes appeared to be prompted by the K(v)7.3 P574S substitution. Computational modelling experiments in CA1 pyramidal cells revealed that the gating changes introduced by the K(v)7.2 D212G increased cell firing frequency, thereby triggering the neuronal hyperexcitability which underlies the observed neonatal epileptic condition.

In this study, the functional consequences of the pharmacological modulation of the M-current (I(KM)) on cytoplasmic Ca(2+) intracellular Ca(2+)concentration ([Ca(2+)](i)) changes and excitatory neurotransmitter release triggered by various stimuli from isolated rat cortical synaptosomes have been investigated. K(v)7.2 immunoreactivity was identified in pre-synaptic elements in cortical slices and isolated glutamatergic cortical synaptosomes. In cerebrocortical synaptosomes exposed to 20 mM [K(+)](e), the I(KM) activator retigabine (RT, 10 microM) inhibited [(3)H]D-aspartate ([(3)H]D-Asp) release and caused membrane hyperpolarization; both these effects were prevented by the I(KM) blocker XE-991 (20 microM). The I(KM) activators RT (0.1-30 microM), flupirtine (10 microM) and BMS-204352 (10 microM) inhibited 20 mM [K(+)](e)-induced synaptosomal [Ca(2+)](i) increases; XE-991 (20 microM) abolished RT-induced inhibition of depolarization-triggered [Ca(2+)](i) transients. The P/Q-type voltage-sensitive Ca(2+)channel (VSCC) blocker omega-agatoxin IVA prevented RT-induced inhibition of depolarization-induced [Ca(2+)](i) increase and [(3)H]D-Asp release, whereas the N-type blocker omega-conotoxin GVIA failed to do so. Finally, 10 microM RT did not modify the increase of [Ca(2+)](i) and the resulting enhancement of [(3)H]D-Asp release induced by [Ca(2+)](i) mobilization from intracellular stores, or by store-operated Ca(2+)channel activation. Collectively, the present data reveal that the pharmacological activation of I(KM) regulates depolarization-induced [(3)H]D-Asp release from cerebrocortical synaptosomes by selectively controlling the changes of [Ca(2+)](i) occurring through P/Q-type VSCCs.