Mutations in

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.H eteromeric assembly of K V 7.2 (KCNQ2) and K V 7.3 (KCNQ3) voltage-dependent K + channel subunits underlies the M-current (I KM ) (1), a slowly activating and deactivating K + neuronal current that regulates excitability in the subthreshold range for action potential (AP) generation (2, 3) and is also involved in network oscillation and synchronization control (4).Mutations in K V 7.2 (5, 6) and, more rarely, K V 7.3 (7) genes are responsible for benign familial neonatal seizures (BFNS), a rare, autosomal-dominant epilepsy of newborns characterized by recurrent seizures that begin in the very first days of life in otherwise healthy newborns and remit after a few weeks or months; BFNS-affected individuals mostly display normal interictal EEG, neuroimaging findings, and psychomotor development.More recently, K V 7.2 mutations have been described in neonates affected with pharmacoresistant seizures with psychomotor retardation, suppression-burst pattern at EEG, and distinct neuroradiological features, thus defining a so-called "K V 7.2 encephalopathy" (8), as well as in children with Ohtahara syndrome or early infantile epileptic encephalopathy with supp...

No abstract

BACKGROUND AND PURPOSEPalmitoylethanolamide (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 ([Ca 2+ ]i) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARa and of TRPV1 channels in PEA-induced effects was also studied. [Ca 2+ ]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. EXPERIMENTAL APPROACH KEY RESULTSIn F11 cells, PEA (1-30 mM) dose-dependently increased [Ca 2+ ]i. The TRPV1 antagonists capsazepine (1 mM) and SB-366791 (1 mM), as well as the PPARa antagonist GW-6471 (10 mM), inhibited PEA-induced [Ca 2+ ]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 PPARa. 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 CAPand BK-induced [Ca 2+ ]i transients, as well as CAP-induced TRPV1 activation. CONCLUSIONS AND IMPLICATIONSActivation of PPARa and TRPV1 channels, rather than of cannabinoid receptors, largely mediate PEA-induced [Ca 2+ ]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.

SUMMARY Variants in KCNQ2 encoding for Kv7.2 neuronal K+ channel subunits lead to a spectrum of neonatal-onset epilepsies ranging from self-limiting forms to severe epileptic encephalopathy. Most KCNQ2 pathogenic variants cause loss-of-function, whereas few increase channel activity (gain-of-function). We herein provide evidence for a new phenotypic and functional profile in KCNQ2 related epilepsy: infantile spasms without prior neonatal seizures associated to a gain-of-function gene variant. Via an international registry, we identified four unrelated patients with de novo heterozygous KCNQ2 c.593G>A, p.R198Q variants. All were born at term and discharged home without seizures or concern of encephalopathy, but developed infantile spasms with hypsarrhythmia (or modified hypsarrhythmia) between the ages of 4-6 months. At last follow up (ages 3-11 years), all patients were seizure-free and had severe developmental delay. In vitro experiments showed that Kv7.2 R198Q subunits shifted current activation gating to hyperpolarized potentials, indicative of gain-of-function; in neurons, Kv7.2 and Kv7.2 R198Q subunits similarly populated the axon initial segment, suggesting that gating changes rather than altered sub-cellular distribution contribute to disease molecular pathogenesis. We conclude that KCNQ2 R198Q is a model for a new subclass of KCNQ2 variants causing infantile spasms and encephalopathy, without preceding neonatal seizures.