Simulating the ion permeation and ion selection for a eukaryotic voltage-gated sodium channel NaVPaS.
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ABSTRACT:
SUBMITTER: Zhang J
PROVIDER: S-EPMC5966359 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Recent determination of the crystal structures of bacterial voltage-gated sodium (NaV) channels have raised hopes that modeling of the mammalian counterparts could soon be achieved. However, there are substantial differences between the pore domains of the bacterial and mammalian NaV channels, which necessitates careful validation of mammalian homology models constructed from the bacterial NaV structures. Such a validated homology model for the NaV1.4 channel was constructed recently using the extensive mutagenesis data available for binding of μ-conotoxins. Here we use this NaV1.4 model to study the ion permeation mechanism in mammalian NaV channels. Linking of the DEKA residues in the selectivity filter with residues in the neighboring domains is found to be important for keeping the permeation pathway open. Molecular dynamics simulations and potential of mean force calculations reveal that there is a binding site for a Na+ ion just inside the DEKA locus, and 1-2 Na+ ions can occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting that inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The NaV1.4 model also provides a good description of the observed Na+/K+ selectivity.
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Project description:The recent determination of cryo-EM structures of voltage-gated sodium (Nav) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Nav pore remains limited. In this work, we performed atomistic molecular dynamics (MD) simulations to study the structural features of various neuronal Nav channels based on homology modeling of the cryo-EM structure of the human Nav1.4 channel and, in addition, on the recently resolved configuration for Nav1.2. In particular, single Na+ permeation events during standard MD runs suggest that the ion resides in the inner part of the Nav selectivity filter (SF). On-the-fly free energy parametrization (OTFP) temperature-accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na+ translocation through the SF of the homology-based Nav1.2 model and the cryo-EM Nav1.2 structure, with different realizations of the DEKA filter domain. These additional simulations revealed distinct mechanisms for single and double Na+ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. Moreover, the configurations of the ions in the SF corresponding to the metastable states of the FESs are specific for each SF motif. Overall, the description of these mechanisms gives us new insights into ion conduction in human Nav cryo-EM-based and cryo-EM configurations that could advance understanding of these systems and how they differ from potassium and bacterial Nav channels.
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Project description:Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits β1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the β1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.
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Project description:Background: Endometrial cancer is the most common gynecologic malignancy in women in the developed countries. Despite recent progress in functional characterization of voltage-gated sodium channel (Nav) in multiple cancers, very little was known about the expression of Nav in human endometrial cancer. The present study sought to determine the role of Nav and molecular nature of this channel in the endometrial cancer. Methods: PCR approach was introduced to determine expression level of Nav subunits in endometrial cancer specimens. Pharmacological agents were used to investigate Nav function in endometrial cancer cells. Flow cytometry were used to test cancer apoptosis, and invasion assays were applied to test tumor metastasis. Results: Transcriptional levels of the all Nav α and β subunits were determined by real time-PCR in endometrial cancer with pair tissues of carcinoma and adjacent nonneoplastic tissue, Nav1.7 was the most highly expressed Nav subtype in endometrial cancer tissues. Nav1.7 level was closely associated with tumor size, local lymph node metastasis, and 5-year and 10-year survival ratio. Inhibition of this channel by Nav1.7 blocker PF-05089771, promoted cancer apoptosis and attenuated cancer cell invasion. Conclusion: These results establish a relationship between voltage-gated sodium channel protein and endometrial cancer, and suggest that Nav1.7 is a potential prognostic biomarker and could serve as a novel therapeutic target for endometrial cancer.
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Project description:µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.
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Project description:It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.
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Project description:Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD ∼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
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Project description:The voltage-gated sodium channel Nav1.8 mediates the tetrodotoxin-resistant (TTX-R) Na+ current in nociceptive primary sensory neurons, which has an important role in the transmission of painful stimuli. Here, we describe the functional modulation of the human Nav1.8 α-subunit in Xenopus oocytes by auxiliary β subunits. We found that the β3 subunit down-regulated the maximal Na+ current amplitude and decelerated recovery from inactivation of hNav1.8, whereas the β1 and β2 subunits had no such effects. The specific regulation of Nav1.8 by the β3 subunit constitutes a potential novel regulatory mechanism of the TTX-R Na+ current in primary sensory neurons with potential implications in chronic pain states. In particular, neuropathic pain states are characterized by a down-regulation of Nav1.8 accompanied by increased expression of the β3 subunit. Our results suggest that these two phenomena may be correlated, and that increased levels of the β3 subunit may directly contribute to the down-regulation of Nav1.8. To determine which domain of the β3 subunit is responsible for the specific regulation of hNav1.8, we created chimeras of the β1 and β3 subunits and co-expressed them with the hNav1.8 α-subunit in Xenopus oocytes. The intracellular domain of the β3 subunit was shown to be responsible for the down-regulation of maximal Nav1.8 current amplitudes. In contrast, the extracellular domain mediated the effect of the β3 subunit on hNav1.8 recovery kinetics.
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Project description:Voltage-gated sodium (NaV) channels initiate action potentials. Fast inactivation of NaV channels, mediated by an Ile-Phe-Met motif, is crucial for preventing hyperexcitability and regulating firing frequency. Here we present cryo-electron microscopy structure of NaVEh from the coccolithophore Emiliania huxleyi, which reveals an unexpected molecular gating mechanism for NaV channel fast inactivation independent of the Ile-Phe-Met motif. An N-terminal helix of NaVEh plugs into the open activation gate and blocks it. The binding pose of the helix is stabilized by multiple electrostatic interactions. Deletion of the helix or mutations blocking the electrostatic interactions completely abolished the fast inactivation. These strong interactions enable rapid inactivation, but also delay recovery from fast inactivation, which is ~160-fold slower than human NaV channels. Together, our results provide mechanistic insights into fast inactivation of NaVEh that fundamentally differs from the conventional local allosteric inhibition, revealing both surprising structural diversity and functional conservation of ion channel inactivation.
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Project description:Scn2a encodes the voltage-gated sodium channel NaV1.2, a main mediator of neuronal action potential firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability, resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability, contributing to autism. However, this paradigm does not explain why ∼20%-30% of individuals with NaV1.2 deficiency still develop seizures. Here, we report the counterintuitive finding that severe NaV1.2 deficiency results in increased neuronal excitability. Using a NaV1.2-deficient mouse model, we show enhanced intrinsic excitability of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in Scn2a-related seizures. This increased excitability is autonomous and reversible by genetic restoration of Scn2a expression in adult mice. RNA sequencing reveals downregulation of multiple potassium channels, including KV1.1. Correspondingly, KV channel openers alleviate the hyperexcitability of NaV1.2-deficient neurons. This unexpected neuronal hyperexcitability may serve as a cellular basis underlying NaV1.2 deficiency-related seizures.
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