Search

Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.

Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel Na(V)1.1 causes Dravets syndrome, a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravets syndrome are poorly understood. Here we report that mice with Scn1a haploinsufficiency exhibit hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive to Scn1a(+/-) mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of Na(V)1.1 channels in forebrain interneurons is sufficient to cause these behavioural and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABA(A) receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravets syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for Na(V)1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravets syndrome.

Voltage-gated calcium ([Ca.sub.V]) channels catalyse rapid, highly selective influx of [Ca.sup.2+] into cells despite a 70-fold higher extracellular concentration of [Na.sup.+] .How [Ca.sub.V] channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial [Na.sub.V] channel [Na.sub.V]Ab. Our results reveal interactions of hydrated [Ca.sup.2+] with two high-affinity [Ca.sup.2+] -binding sites followed by a third lower-affinity site that would coordinate [Ca.sup.2+] as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining [Ca.sup.2+] selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations [Cd.sup.2+] and [Mn.sup.2+], with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated [Ca.sup.2+] ions bound in the selectivity filter and supports a 'knock-off' mechanism of ion permeation through a stepwisebinding process. The multi-ion selectivity filter of our [Ca.sub.V]Ab model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate [Ca.sub.V] channels., [Ca.sup.2+] ions flow through [Ca.sub.V] channels at a rate of ~[10.sup.6] ions [s.sup.-1], yet [Na.sup.+] conductance is >500-fold lower (1). Such high-fidelity, high-throughput [Ca.sub.V] channel performance is important in regulating [...]