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Here, we report the discovery of a novel anticonvulsant drug with a molecular organization based on the unique scaffold of rufinamide, an anti-epileptic compound used in a clinical setting to treat severe epilepsy disorders such as Lennox-Gastaut syndrome. Although accumulating evidence supports a working mechanism through voltage-gated sodium (Nav) channels, we found that a clinically relevant rufinamide concentration inhibits human (h)Nav1.1 activation, a distinct working mechanism among anticonvulsants and a feature worth exploring for treating a growing number of debilitating disorders involving hNav1.1. Subsequent structure–activity relationship experiments with related N-benzyl triazole compounds on four brain hNav channel isoforms revealed a novel drug variant that (1) shifts hNav1.1 opening to more depolarized voltages without further alterations in the gating properties of hNav1.1, hNav1.2, hNav1.3, and hNav1.6; (2) increases the threshold to action potential initiation in hippocampal neurons; and (3) greatly reduces the frequency of seizures in three animal models. Altogether, our results provide novel molecular insights into the rational development of Nav channel-targeting molecules based on the unique rufinamide scaffold, an outcome that may be exploited to design drugs for treating disorders involving particular Nav channel isoforms while limiting adverse effects.

GABA A receptors shape synaptic transmission by modulating Cl − conductance across the cell membrane. Remarkably, animal toxins that specifically target GABA A receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABA A receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABA A receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α + /β − interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABA A receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.

SUMMARY Better understanding of the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here, we use RNA sequencing, cell imaging, and lineage tracing of mouse and human in vitro NSCs and monkey brain sections to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs develop from pluripotency in vitro , they transition through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines vary in these early patterning states, leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analyses of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

SUMMARYBetter understanding the progression of neural stem cells (NSCs) in the developing cerebral cortex is important for modeling neurogenesis and defining the pathogenesis of neuropsychiatric disorders. Here we used RNA-sequencing, cell imaging and lineage tracing of mouse and human in vitro NSCs to model the generation of cortical neuronal fates. We show that conserved signaling mechanisms regulate the acute transition from proliferative NSCs to committed glutamatergic excitatory neurons. As human telencephalic NSCs developed from pluripotency in vitro, they first transitioned through organizer states that spatially pattern the cortex before generating glutamatergic precursor fates. NSCs derived from multiple human pluripotent lines varied in these early patterning states leading differentially to dorsal or ventral telencephalic fates. This work furthers systematic analysis of the earliest patterning events that generate the major neuronal trajectories of the human telencephalon.

electrophysiology in cultured rat striatal neurons and transfected HEK 293 cells. We determined that 79 53% of cultured cells endogenously expressed b3, and 94% of neurons gave tonic-level GABA-evoked currents blocked by phasic concentrations of dopamine (0.1-10 mM). Inhibition was recapitulated in HEK 293 cells transfected with a1b3, a1b3d or a1b2g2 subunits, but we instead observed potentiation in a1b3g2 and a5b3g2. Surprisingly, dopamine (1 mM) evoked rapid currents in a1b2g2, a1b3g2 and a5b3g2 in the absence of GABA. In a1b3(H267A)g2 (a1Zb3g2) receptors insensitive to trace Zn2þ inhibition, we found that other biogenic amines evoked comparatively smaller currents than DA. When the ratio of a1 was increased or we mutated a critical Zb3 tyrosine residue (Y62L), relative dopamine responses decreased. Furthermore, dopamine activity was retained but GABA activity was drastically reduced in Zb3g2 receptors, while Zb3 or g2 alone did not elicit currents. Finally, dopamine currents were picrotoxin-but not bicuculline-or gabazinesensitive. Taken together, dopamine at phasic levels is a GABAA allosteric modulator that may inhibit tonic GABA in prototypical a-containing GABAA receptors. However, at b/g-containing receptors that do not need a subunits, dopamine acts as a positive allosteric modulator.

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channels has allowed us to learn much about ion conduction mechanisms, but our understanding of their mammalian counterparts remains limited. A model of the human Na v 1.2 channel has been constructed by grafting residues of its selectivity filter and external vestibular region onto the bacterial Na v Rh channel. Multi-microsecond fully atomistic simulations, using the DE Shaw Anton supercomputer, capture long time-scale ion and protein movements associated with ion permeation. We observe Na þ knock-on conduction facilitated by low energy multiple carboxylate-Na þ complexes, akin to the bacterial channels. These complexes draw carboxylates from both the DEKA and vestibular EEDD rings. We observe that lysine, when charged, actively participates in ion movements, with the critical step involving the formation of a complex that collectively binds Na þ and lysine in a high field strength site. In contrast, multiple K þ complexes are disfavored, and K þlysine complexes nonexistent. Instead, lysine acts as an plug that attenuates the flow of K þ ions. These mechanisms, controlled by different Na þ and K þ affinities for high field strength complexes, helps explain Na þ selectivity across all sodium channels.