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BackgroundMalformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants inGRIN1orGRIN2B, genes which encode for subunits of the N-methyl-D-aspartate receptor (NMDAR). The aim of this study was to further define the phenotypic spectrum of NMDAR-related MCDs.MethodsWe report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants inGRIN1are presented. We report the clinical and neuropathological features of one additional individual with homozygous pathogenic variants inGRIN1.ResultsHeterozygous variants inGRIN1andGRIN2Bwere associated with overlapping severe clinical and imaging features, including global developmental delay, epilepsy, diffuse dysgyria, dysmorphic basal ganglia and hippocampi. Neuropathological examination in two fetuses with heterozygousGRIN1variants suggests that proliferation as well as radial and tangential neuronal migration are impaired. In addition, we show that neuronal migration is also impaired by homozygousGRIN1variants in an individual with microcephaly with simplified gyral pattern.ConclusionThese findings expand our understanding of the clinical and imaging features of the ‘NMDARopathy’ spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.

Background Mutations in the NMDA receptor are known to disrupt glutamatergic signaling crucial for early neurodevelopment, often leading to severe global developmental delay/intellectual disability, epileptic encephalopathy, and cerebral palsy phenotypes. Both seizures and movement disorders can be highly treatment-refractory. Results We describe a targeted ABA n = 1 patient trial with intrathecal MgSO4, rationally designed based on the electrophysiologic properties of this gain of function mutation in the GRIN1 NMDA subunit. Conclusion Although the invasive nature of the trial necessitated a short-term, non-randomized, unblinded intervention, multimodal neurophysiologic monitoring indicated benefit, providing class II evidence in support of intrathecal MgSO4 for select forms of GRIN disorders.

N-methyl-D-aspartate receptors (NMDARs) are tetrameric assemblies of two glutamate N-methyl-D-aspartate receptor subunits, GluN1 and two GluN2, that mediate excitatory synaptic transmission in the central nervous system. Four genes (

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine’s antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Malformations of cortical development (MCDs) have been reported in a subset of patients with pathogenic heterozygous variants inWe report the clinical, radiological and molecular features of 7 new patients and review data on 18 previously reported individuals with NMDAR-related MCDs. Neuropathological findings for two individuals with heterozygous variants inHeterozygous variants inThese findings expand our understanding of the clinical and imaging features of the 'NMDARopathy' spectrum and contribute to our understanding of the likely underlying pathogenic mechanisms leading to MCD in these patients.

N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

Background and purpose Kv 7.4 and Kv 7.5 channels are regulators of vascular tone. 4-Aminopyridine (4-AP) is considered a broad inhibitor of voltage-gated potassium (KV ) channels, with little inhibitory effect on Kv 7 family members at mmol concentrations. However, the effect of 4-AP on Kv 7 channels has not been systematically studied. The aim of this study was to investigate the pharmacological activity of 4-AP on Kv 7.4 and Kv 7.5 channels and characterize the effect of 4-AP on rat resistance arteries. Experimental approach Voltage clamp experiments were performed on Xenopus laevis oocytes injected with cRNA encoding KCNQ4 or KCNQ5, HEK cells expressing Kv 7.4 channels and on rat, freshly isolated mesenteric artery smooth muscle cells. The effect of 4-AP on tension, membrane potential, intracellular calcium and pH was assessed in rat mesenteric artery segments. Key results 4-AP increased the Kv 7.4-mediated current in oocytes and HEK cells but did not affect Kv 7.5 current. 4-AP also enhanced native mesenteric artery myocyte K+ current at sub-mmol concentrations. When applied to NA-preconstricted mesenteric artery segments, 4-AP hyperpolarized the membrane, decreased [Ca2+ ]i and caused concentration-dependent relaxations that were independent of 4-AP-mediated changes in intracellular pH. Application of the Kv 7 channel blocker XE991 and BKCa channel blocker iberiotoxin attenuated 4-AP-mediated relaxation. 4-AP also inhibited the NA-mediated signal transduction to elicit a relaxation. Conclusions and implications These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of Kv 7.4 and BKCa channels and attenuating NA-mediated signalling.

NMDA receptors (NMDAR) play crucial roles in excitatory synaptic transmission. Rare variants ofGRIN2A, which encodes the GluN2A NMDAR subunit, are associated with several intractable neurodevelopmental disorders, including developmental and epileptic encephalopathy (DEE). Ade novomissense variant, p.Ser644Gly (c.1930A>G), was identified in a child with DEE, andGrin2aknockin mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibit altered hippocampal morphology at two weeks of age, and homozygotes exhibit lethal tonic-clonic seizures in the third week. Heterozygous adult mice display a variety of distinct features, including resistance to electrically induced partial seizures, as well as hyperactivity and repetitive and reduced anxiety behaviors. Multielectrode recordings of mutant neuronal networks reveal hyperexcitability and altered bursting and synchronicity. When expressed in heterologous cells, mutant receptors exhibit enhanced NMDAR agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. Consistent with these observations, NMDAR-mediated synaptic currents in hippocampal slices from mutant mice show a prolonged deactivation time course. Standard antiepileptic drug monotherapy was ineffective in the patient, but combined treatment of NMDAR antagonists with antiepileptic drugs substantially reduced the seizure burden albeit without appreciable developmental improvement. Chronic treatment of homozygous mutant mouse pups with NMDAR antagonists delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of modeling severe neurodevelopmental seizure disorders using multiple experimental modalities and suggest their extended utility in identifying and evaluating new therapies.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2+/−G301R) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca2+and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2+/−G301Rand WT showed similar contractile responses. The Atp1a2+/−G301RMCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca2+change was smaller than in WT. The enhanced constriction of Atp1a2+/−G301RMCA was associated with increased cSrc activation, stronger sensitization to [Ca2+]iand increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2+/−G301Rmice had reduced resting blood flow through MCA in comparison with WT mice . FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca2+, which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine’s antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED(50) of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Familial hemiplegic migraine type 2 (FHM2) is associated with inherited point-mutations in the Na,K-ATPase α2 isoform, including G301R mutation. We hypothesized that this mutation affects specific aspects of vascular function, and thus compared cerebral and systemic arteries from heterozygote mice bearing the G301R mutation (Atp1a2(+/−G301R)) with wild type (WT). Middle cerebral (MCA) and mesenteric small artery (MSA) function was compared in an isometric myograph. Cerebral blood flow was assessed with Laser speckle analysis. Intracellular Ca(2+) and membrane potential were measured simultaneously. Protein expression was semi-quantified by immunohistochemistry. Protein phosphorylation was analysed by Western blot. MSA from Atp1a2(+/−G301R) and WT showed similar contractile responses. The Atp1a2(+/−G301R) MCA constricted stronger to U46619, endothelin and potassium compared to WT. This was associated with an increased depolarization, although the Ca(2+) change was smaller than in WT. The enhanced constriction of Atp1a2(+/−G301R) MCA was associated with increased cSrc activation, stronger sensitization to [Ca(2+)](i) and increased MYPT1 phosphorylation. These differences were abolished by cSrc inhibition. Atp1a2(+/−G301R) mice had reduced resting blood flow through MCA in comparison with WT mice. FHM2-associated mutation leads to elevated contractility of MCA due to sensitization of the contractile machinery to Ca(2+), which is mediated via Na,K-ATPase/Src-kinase/MYPT1 signalling.

Interactions between perivascular tissue (PVT) and the vascular wall modify artery tone and contribute to local blood flow regulation. Using isometric myography, fluorescence microscopy, membrane potential recordings and phosphospecific immunoblotting, we investigated the cellular mechanisms by which PVT affects constriction and relaxation of rat coronary septal arteries. PVT inhibited vasoconstriction to thromboxane, serotonin and α1 -adrenergic stimulation but not to depolarization with elevated extracellular [K(+) ]. When PVT was wrapped around isolated arteries or placed at the bottom of the myograph chamber, a smaller yet significant inhibition of vasoconstriction was observed. Resting membrane potential, depolarization to serotonin or thromboxane stimulation, and resting and serotonin-stimulated vascular smooth muscle [Ca(2+) ]-levels were unaffected by PVT. Serotonin-induced vasoconstriction was almost abolished by rho-kinase inhibitor Y-27632 and modestly reduced by protein kinase C inhibitor bisindolylmaleimide X. PVT reduced phosphorylation of myosin phosphatase targeting subunit (MYPT) at Thr850 by ∼40% in serotonin-stimulated arteries but had no effect on MYPT-phosphorylation in arteries depolarized with elevated extracellular [K(+) ]. The net anti-contractile effect of PVT was accentuated after endothelial denudation. PVT also impaired vasorelaxation and endothelial Ca(2+) responses to cholinergic stimulation. Methacholine-induced vasorelaxation was mediated by NO and H2 S, and particularly the H2 S-dependent (dl-propargylglycine- and XE991-sensitive) component was attenuated by PVT. Vasorelaxation to NO- and H2 S-donors was maintained in arteries with PVT. In conclusion, cardiomyocyte-rich PVT surrounding coronary arteries releases diffusible factors that reduce rho-kinase-dependent smooth muscle Ca(2+) sensitivity and endothelial Ca(2+) responses. These mechanisms inhibit agonist-induced vasoconstriction and endothelium-dependent vasorelaxation and suggest new signalling pathways for metabolic regulation of blood flow.

Voltage-gated sodium channels (VGSC) play a key role for initiating action potentials (AP) in excitable cells. VGSC in human lymphatic vessels have not been investigated. In the present study, we report the electrical activity and APs of small human lymphatic collecting vessels, as well as mRNA expression and function of VGSC in small and large human lymphatic vessels. The VGSC blocker TTX inhibited spontaneous contractions in six of 10 spontaneously active vessels, whereas ranolazine, which has a narrower VGSC blocking profile, had no influence on spontaneous activity. TTX did not affect noradrenaline-induced contractions. The VGSC opener veratridine induced contractions in a concentration-dependent manner (0.1-30 μm) eliciting a stable tonic contraction and membrane depolarization to -18 ± 0.6 mV. Veratridine-induced depolarizations and contractions were reversed ∼80% by TTX, and were dependent on Ca(2+) influx via L-type calcium channels and the sodium-calcium exchanger in reverse mode. Molecular analysis determined NaV 1.3 to be the predominantly expressed VGSC isoform. Electrophysiology of mesenteric lymphatics determined the resting membrane potential to be -45 ± 1.7 mV. Spontaneous APs were preceded by a slow depolarization of 5.3 ± 0.6 mV after which a spike was elicited that almost completely repolarized before immediately depolarizing again to plateau. Vessels transiently hyperpolarized prior to returning to the resting membrane potential. TTX application blocked APs. We have shown that VGSC are necessary for initiating and maintaining APs and spontaneous contractions in human lymphatic vessels and our data suggest the main contribution from comes NaV 1.3. We have also shown that activation of these channels augments the contractile activity of the vessels.

Background and purpose T16A(inh)-A01, CaCC(inh)-A01 and MONNA are identified as selective inhibitors of the TMEM16A calcium-activated chloride channel (CaCC). The aim of this study was to examine the chloride-specificity of these compounds on isolated resistance arteries in the presence and absence (±) of extracellular chloride. Experimental approach Isolated resistance arteries were maintained in a myograph and tension recorded, in some instances combined with microelectrode impalement for membrane potential measurements or intracellular calcium monitoring using fura-2. Voltage-dependent calcium currents (VDCC) were measured in A7r5 cells with voltage-clamp electrophysiology using barium as a charge carrier. Key results Rodent arteries preconstricted with noradrenaline or U46619 were concentration-dependently relaxed by T16A(inh) -A01 (0.1-10 μM): IC50 and maximum relaxation were equivalent in ±chloride (30 min aspartate substitution) and the T16A(inh) -A01-induced vasorelaxation ±chloride were accompanied by membrane hyperpolarization and lowering of intracellular calcium. However, agonist concentration-response curves ±chloride, with 10 μM T16A(inh) -A01 present, achieved similar maximum constrictions although agonist-sensitivity decreased. Contractions induced by elevated extracellular potassium were concentration-dependently relaxed by T16A(inh)-A01 ±chloride. Moreover, T16A(inh) -A01 inhibited VDCCs in A7r5 cells in a concentration-dependent manner. CaCC(inh) -A01 and MONNA (0.1-10 μM) induced vasorelaxation ±chloride and both compounds lowered maximum contractility. MONNA, 10 μM, induced substantial membrane hyperpolarization under resting conditions. Conclusions and implications T16A(inh) -A01, CaCC(inh) -A01 and MONNA concentration-dependently relax rodent resistance arteries, but an equivalent vasorelaxation occurs when the transmembrane chloride gradient is abolished with an impermeant anion. These compounds therefore display poor selectivity for TMEM16A and inhibition of CaCC in vascular tissue in the concentration range that inhibits the isolated conductance.

Figure S1. Vasocontraction is attenuated in breast cancer feed arteries compared to matched control arteries under acidic conditions. Concentration-dependent contractions in response to norepinephrine (A), endothelin-1 (B), and elevated [K+]o (C) at pHo 6.8 (n = 8). For norepinephrine and K+, contractions elicited in the same arteries at pHo 7.4 are shown for comparison. Because of slow relaxation of arteries after washout of endothelin-1, vasocontraction to endothelin-1 was only tested at one level of pHo for each artery. Experiments with elevated [K+]o were performed in presence of 1 μmol/L phentolamine in order to avoid effects of norepinephrine released from sympathetic nerve endings in response to depolarization. Curves are results of least-squares fits to sigmoidal functions and compared using extra-sum-of-squares F tests. **P

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We tested here the hypothesis that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance, and synchronized [Ca2+]itransients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2, and by an inhibitor of the Na-K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity whereas pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation.