CryoEM micrographs of ProTx2-bound Nav1.7 VSD2-NavAb chimeric channel
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ABSTRACT:
SUBMITTER: Alexis Lawrence Rohou
PROVIDER: EMPIAR-10261 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.
| S-EPMC3750134 | biostudies-literature
Project description: Not available
| EMPIAR-11794 | biostudies-other
Project description: Not available
| EMPIAR-11790 | biostudies-other
Project description:Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.
| S-EPMC3674497 | biostudies-literature
Project description:BACKGROUND: Voltage-gated sodium channel Nav1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic neurons within the peripheral nervous system. Homozygous or compound heterozygous loss-of-function mutations in SCN9A, the gene which encodes Nav1.7, cause congenital insensitivity to pain (CIP) accompanied by anosmia. Global knock-out of Nav1.7 in mice is neonatal lethal reportedly from starvation, suggesting anosmia. These findings led us to hypothesize that Nav1.7 is the main sodium channel in the peripheral olfactory sensory neurons (OSN, also known as olfactory receptor neurons). METHODS: We used multiplex PCR-restriction enzyme polymorphism, in situ hybridization and immunohistochemistry to determine the identity of sodium channels in rodent OSNs. RESULTS: We show here that Nav1.7 is the predominant sodium channel transcript, with low abundance of other sodium channel transcripts, in olfactory epithelium from rat and mouse. Our in situ hybridization data show that Nav1.7 transcripts are present in rat OSNs. Immunostaining of Nav1.7 and Nav1.6 channels in rat shows a complementary accumulation pattern with Nav1.7 in peripheral presynaptic OSN axons, and Nav1.6 primarily in postsynaptic cells and their dendrites in the glomeruli of the olfactory bulb within the central nervous system. CONCLUSIONS: Our data show that Nav1.7 is the dominant sodium channel in rat and mouse OSN, and may explain anosmia in Nav1.7 null mouse and patients with Nav1.7-related CIP.
| S-EPMC3101130 | biostudies-literature
Project description:Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39?µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53?µM], also in a dose-dependent manner. Veratridine (75?µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64?±?0.75?mV to -28.14?±?0.66?mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39?±?0.39?mV to -73.78?±?0.5?mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.
| S-EPMC6289357 | biostudies-literature
Project description:BackgroundGain-of-function mutations in SCN9A gene that encodes the voltage-gated sodium channel NaV1.7 have been associated with a wide spectrum of painful syndromes in humans including inherited erythromelalgia, paroxysmal extreme pain disorder and small fibre neuropathy. These mutations change the biophysical properties of NaV1.7 channels leading to hyperexcitability of dorsal root ganglion nociceptors and pain symptoms. There is a need for better understanding of how gain-of-function mutations alter the atomic structure of Nav1.7.ResultsWe used homology modeling to build an atomic model of NaV1.7 and a network-based theoretical approach, which can predict interatomic interactions and connectivity arrangements, to investigate how pain-related NaV1.7 mutations may alter specific interatomic bonds and cause connectivity rearrangement, compared to benign variants and polymorphisms. For each amino acid substitution, we calculated the topological parameters betweenness centrality (B ct ), degree (D), clustering coefficient (CC ct ), closeness (C ct ), and eccentricity (E ct ), and calculated their variation (Δ value = mutant value -WT value ). Pathogenic NaV1.7 mutations showed significantly higher variation of |ΔB ct | compared to benign variants and polymorphisms. Using the cut-off value ±0.26 calculated by receiver operating curve analysis, we found that ΔB ct correctly differentiated pathogenic NaV1.7 mutations from variants not causing biophysical abnormalities (nABN) and homologous SNPs (hSNPs) with 76% sensitivity and 83% specificity.ConclusionsOur in-silico analyses predict that pain-related pathogenic NaV1.7 mutations may affect the network topological properties of the protein and suggest |ΔB ct | value as a potential in-silico marker.
| S-EPMC5324268 | biostudies-literature
Project description:Voltage-gated sodium channel 1.7 (Nav1.7) remains one of the most promising drug targets for pain relief. In the current study, we conducted a high-throughput screening of natural products in our in-house compound library to discover novel Nav1.7 inhibitors, then characterized their pharmacological properties. We identified 25 naphthylisoquinoline alkaloids (NIQs) from Ancistrocladus tectorius to be a novel type of Nav1.7 channel inhibitors. Their stereostructures including the linkage modes of the naphthalene group at the isoquinoline core were revealed by a comprehensive analysis of HRESIMS, 1D, and 2D NMR spectra as well as ECD spectra and single-crystal X-ray diffraction analysis with Cu Kα radiation. All the NIQs showed inhibitory activities against the Nav1.7 channel stably expressed in HEK293 cells, and the naphthalene ring in the C-7 position displayed a more important role in the inhibitory activity than that in the C-5 site. Among the NIQs tested, compound 2 was the most potent with an IC50 of 0.73 ± 0.03 µM. We demonstrated that compound 2 (3 µM) caused dramatical shift of steady-state slow inactivation toward the hyperpolarizing direction (V1/2 values were changed from -39.54 ± 2.77 mV to -65.53 ± 4.39 mV, which might contribute to the inhibition of compound 2 against the Nav1.7 channel. In acutely isolated dorsal root ganglion (DRG) neurons, compound 2 (10 μM) dramatically suppressed native sodium currents and action potential firing. In the formalin-induced mouse inflammatory pain model, local intraplantar administration of compound 2 (2, 20, 200 nmol) dose-dependently attenuated the nociceptive behaviors. In summary, NIQs represent a new type of Nav1.7 channel inhibitors and may act as structural templates for the following analgesic drug development.
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Project description:Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxyl-terminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.
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