Local anesthetic inhibition of a bacterial sodium channel

101

199

Voltage-gated Na + channels play an essential role in electrical signaling in the nervous system and are key pharmacological targets for a range of disorders. The recent solution of X-ray structures for the bacterial channel Na v Ab has provided an opportunity to study functional mechanisms at the atomic level. This channel's selectivity filter exhibits an EEEE ring sequence, characteristic of mammalian Ca 2+ , not Na + , channels. This raises the fundamentally important question: just what makes a Na + channel conduct Na + ions? Here we explore ion permeation on multimicrosecond timescales using the purpose-built Anton supercomputer. We isolate the likely protonation states of the EEEE ring and observe a striking flexibility of the filter that demonstrates the necessity for extended simulations to study conduction in this channel. We construct free energy maps to reveal complex multi-ion conduction via knock-on and "pass-by" mechanisms, involving concerted ion and glutamate side chain movements. Simulations in mixed ionic solutions reveal relative energetics for Na + , K + , and Ca 2+ within the pore that are consistent with the modest selectivity seen experimentally. We have observed conformational changes in the pore domain leading to asymmetrical collapses of the activation gate, similar to proposed inactivated structures of Na v Ab, with helix bending involving conserved residues that are critical for slow inactivation. These structural changes are shown to regulate access to fenestrations suggested to be pathways for lipophilic drugs and provide deeper insight into the molecular mechanisms connecting drug activity and slow inactivation.

mentioning

confidence: 99%

mentioning

confidence: 99%

Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel

Boiteux

Vorobyov

Allen

2014

101

199

“…Mutations in human sodium channels (hNa v s) have been linked to channelopathies such as epilepsy, cardiac arrhythmia, and chronic pain syndromes; consequently sodium channel blockers have been developed as anticonvulsant, antiarrhythmic, and local anesthetic drugs (7)(8)(9)(10). Several eukaryotic calcium channel blocker drugs have previously been found to bind and block prokaryotic sodium channels (11)(12)(13).…”

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confidence: 99%

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism

Bagnéris

DeCaen

Naylor

et al. 2014

127

168

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Na v 1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.crystal structure | pharmacology

“…These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac. The local anesthetic binding site identified in the central cavity of the mammalian Na V 1.2 channel, which mediates open channel block, is partially conserved in NaChBac (37,38). A recent molecular dynamics (MD) modeling study found that isoflurane, which inhibits NaChBac (15), interacts with multiple regions of this channel, including the pore, the selectivity filter, and the S4-S5 linker/S6 interface (39).…”

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confidence: 99%

“…Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15,37). These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac.…”

mentioning

confidence: 99%

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

Barber

Carnevale

Klein

et al. 2014

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...