Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

McCusker, Emily C.; Bagnéris, Claire; Naylor, C.E.; Cole, A.R.; D’Avanzo, Nazzareno; Nichols, Colin G.; Wallace, B.A.

doi:10.1038/ncomms2077

Cited by 272 publications

(359 citation statements)

References 35 publications

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“…The differences that enabled the improvement in resolution were small but manifold, as is often the case for membrane proteins and included such things as very minor changes to the solubilisation and thrombin cleavage procedures and small changes to the protein and crystal handling procedures. As previously noted (McCusker et al , 2012), we were unable to grow any crystals under any similar conditions in the absence of sodium ions or when the sodium salts were replaced with other monovalent salts (such as lithium) in the crystallisation condition. Large crystals (above 100 μm) were soaked with 5 and 50 mM ion solutions (made using 100 mM and 1 M stock solutions containing 100% DMSO for cadmium (II) chloride, thallium (I) nitrate, manganese (II) chloride, silver (I) nitrate, or 10 mM Tris, 100 mM NaCl, 0.52% Hega10 [gel filtration buffer] for barium (II) acetate, lithium (I) chloride and calcium (II) chloride), with the aim of replacing the sodium ions with other monovalent or divalent ions to enable the detection of anomalous difference signals.…”

Section: Methodssupporting

confidence: 59%

“…The structures of several prokaryotic sodium channels in different functional states (Payandeh et al , 2011, 2012; McCusker et al , 2012; Zhang et al , 2012; Bagnéris et al , 2013, 2014, 2015; Tsai et al , 2013; Shaya et al , 2014) have been solved by X‐ray crystallography or electron microscopy. Functional studies on wild‐type and mutant prokaryotic channels (Ren et al , 2001; Yue et al , 2002; McCusker et al , 2011; Shaya et al , 2011; Tang et al , 2014) have suggested which regions and residues may be important for ion binding, selectivity and translocation within the SF.…”

Section: Introductionmentioning

confidence: 99%

“…Whilst the locations of sodium ions were proposed based on the structure of the closed pore form (Payandeh et al , 2011), no density was seen in the SF in those crystals to indicate any sodium ions were present. Only structures of the open state (McCusker et al , 2012; Bagnéris et al , 2014) pore of NavMs from Magnetococcus marinus have shown any electron density within the SF that might be attributable to sodium ions. The resolution of the first such structure (3.5 Å), however, was too low to determine the details of the sites of the ions in the SF, whilst the latter structures contained channel blocker ligands which interfered with ion binding.…”

Section: Introductionmentioning

confidence: 99%

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Molecular basis of ion permeability in a voltage‐gated sodium channel

et al. 2016

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Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na+ ≈ Li+ ≫ K+, Ca2+, Mg2+) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.

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Section: Methodssupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular basis of ion permeability in a voltage‐gated sodium channel

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“…However, the recent discovery of the smaller, tetrameric bacterial Na + channel family has provided an invaluable tool to characterize the structural features of Na V channels and investigate their interactions with general anesthetic agents at the molecular level (22,23). Several bacterial Na + channels have been crystallized (24)(25)(26)(27). These channels have a classical domain structure in which helices S1-S4 form the voltage sensor domain (VSD), S5 and S6 form the pore, and the S4-S5 linker connects the voltage sensor to the pore domain.…”

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confidence: 99%

Modulation of a voltage-gated Na⁺channel by sevoflurane involves multiple sites and distinct mechanisms

Barber

Carnevale

Klein

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Halogenated inhaled general anesthetic agents modulate voltagegated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltagegated Na + (Na V ) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.anesthesia | MD simulations | anesthetics | membrane proteins G eneral anesthetic agents have been in use for more than 160 y.However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1-4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5-7). Mammalian voltage-gated Na + (Na V ) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8-14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na + channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11,(15)(16)(17)(18). Inhibition of presynaptic Na V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization-one of the endpoints of general anesthesia (14,19,20). Despite the importance of Na V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na V channel modulation by general anesthetic agents...

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“…8-10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11)(12)(13)(14)(15)(16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5,6,(21)(22)(23).…”

mentioning

confidence: 99%

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7

Thomas‐Tran

Bois

2016

Proc. Natl. Acad. Sci. U.S.A.

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Improper function of voltage-gated sodium channels (Na V s), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na V s hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na V through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na V 1.7 (hNa V 1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na V inhibitors.sodium channel | guanidinium toxin | mutant cycle analysis M odulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na V s) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na V 1.1-Na V 1.9, Na x ) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na V s have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1-4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na V isoforms with nanomolar affinity (5-7). Guanidinium toxins inhibit Na + influx through Na V s by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8-10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11-16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5, 6, 21-23)....

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Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Cited by 272 publications

References 35 publications

Molecular basis of ion permeability in a voltage‐gated sodium channel

Molecular basis of ion permeability in a voltage‐gated sodium channel

Modulation of a voltage-gated Na⁺channel by sevoflurane involves multiple sites and distinct mechanisms

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7

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Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Cited by 272 publications

References 35 publications

Molecular basis of ion permeability in a voltage‐gated sodium channel

Molecular basis of ion permeability in a voltage‐gated sodium channel

Modulation of a voltage-gated Na+channel by sevoflurane involves multiple sites and distinct mechanisms

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa V 1.7

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Modulation of a voltage-gated Na⁺channel by sevoflurane involves multiple sites and distinct mechanisms

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7