Structure of the Cardiac Sodium Channel.

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.

Structural basis for the modulation of voltage-gated sodium channels by animal toxins.

Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.

Sodium channel subtypes are differentially localized to pre- and post-synaptic sites in rat hippocampus.

Voltage-gated Na+ channels (Nav ) modulate neuronal excitability, but the roles of the various Nav subtypes in specific neuronal functions such as synaptic transmission are unclear. We investigated expression of the three major brain Nav subtypes (Nav 1.1, Nav 1.2, Nav 1.6) in area CA1 and dentate gyrus of rat hippocampus. Using light and electron microscopy, we found labeling for all three Nav subtypes on dendrites, dendritic spines, and axon terminals, but the proportion of pre- and post-synaptic labeling for each subtype varied within and between subregions of CA1 and dentate gyrus. In the central hilus (CH) of the dentate gyrus, Nav 1.1 immunoreactivity was selectively expressed in presynaptic profiles, while Nav 1.2 and Nav 1.6 were expressed both pre- and post-synaptically. In contrast, in the stratum radiatum (SR) of CA1, Nav 1.1, Nav 1.2, and Nav 1.6 were selectively expressed in postsynaptic profiles. We next compared differences in Nav subtype expression between CH and SR axon terminals and between CH and SR dendrites and spines. Nav 1.1 and Nav 1.2 immunoreactivity was preferentially localized to CH axon terminals compared to SR, and in SR dendrites and spines compared to CH. No differences in Nav 1.6 immunoreactivity were found between axon terminals of CH and SR or between dendrites and spines of CH and SR. All Nav subtypes in both CH and SR were preferentially associated with asymmetric synapses rather than symmetric synapses. These findings indicate selective presynaptic and postsynaptic Nav expression in glutamatergic synapses of CH and SR supporting neurotransmitter release and synaptic plasticity.

Structure of the Nav1.4-ß1 Complex from Electric Eel.

Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the ß1 subunit at 4.0 Å resolution. The immunoglobulin domain of ß1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Nav channel from American cockroach (designated NavPaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSDI, and a carboxy-terminal domain binds to the III-IV linker. The structure of NavPaS establishes an important foundation for understanding function and disease mechanism of Nav and related voltage-gated calcium channels.

Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides.

Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee's sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee's channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.

Benchmarking the stability of human detergent-solubilised voltage-gated sodium channels for structural studies using eel as a reference.

With the ultimate goal of detailed structural analysis of mammalian and particularly human voltage-gated sodium channels (VGSCs), we have investigated the relative stability of human and rat VGSCs and compared them with electric eel VGSC. We found that NaV1.3 from rat was the most stable after detergent solubilisation. The order of stability was rNaV1.3>hNaV1.2>hNaV1.1>hNaV1.6>hNaV1.3>hNaV1.4. However, a comparison with the VGSC from Electrophorus electricus, which is most similar to NaV1.4, shows that the eel VGSC is considerably more stable in detergent than the human VGSCs examined. We conclude that current methods of structural analysis, such as single particle electron cryomicroscopy (cryoEM), may be most usefully targeted to eel VGSC or rNaV1.3, but that structural analysis on the full spectrum of VGSCs, by methods that require greater stability such as crystallisation and X-ray crystallography, will require further stabilisation of the channel.