Céline Boiteux

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

Significance Voltage-gated sodium channels are one of the most fundamental electrical components in the nervous system and are key targets for local anesthesia and therapeutics for neurological and cardiac disorders. We have used multimicrosecond simulations to provide molecular-level descriptions of sodium channel function. We describe an almost barrier-less three-ion conduction mechanism involving competing knock-on and “pass-by” processes, intimately linked to signature glutamate ring protonation and structural isomerizations. These simulations have uncovered a high degree of protein flexibility, with conformational fluctuations in the pore domain involving residues central to slow-type inactivation, leading to gate collapse, helix bending, filter disruption, and changes in lipid-facing fenestrations linked to Nav drug pathways. 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 NavAb 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 Ca2+, 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 Ca2+ 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 NavAb, 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.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel.

Significance Voltage-gated sodium (Nav) channels control neuronal signaling and are key targets for local anesthetics, antiepileptics, and therapeutics for a range of disorders. Multimicrosecond Anton simulations have provided completely unbiased molecular-level views of the interactions of lipophilic drugs with the recently solved bacterial channel, NavAb from Arcobacter butzleri. Newly parameterized benzocaine and phenytoin molecules exhibit different membrane partition coefficients, crossing rates and distributions around the channel, leading to the identification of distinct high- and low-affinity sites. We observe a minimum free energy pathway through membrane-bound fenestrations to a pore-blocking location, or from aqueous solution directly through the (closed) intracellular gate. These observations help explain experimental data and provide insight into Nav inhibition processes that will assist future drug development. Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the “hinged lid”-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water–protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors.

A selectivity filter at the intracellular end of the acid-sensing ion channel pore

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the “GAS belt” in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction. DOI: http://dx.doi.org/10.7554/eLife.24630.001

Comparison of permeation mechanisms in sodium-selective ion channels

Voltage-gated sodium channels are the molecular components of electrical signaling in the body, yet the molecular origins of Na+-selective transport remain obscured by diverse protein chemistries within this family of ion channels. In particular, bacterial and mammalian sodium channels are known to exhibit similar relative ion permeabilities for Na+ over K+ ions, despite their distinct signature EEEE and DEKA sequences. Atomic-level molecular dynamics simulations using high-resolution bacterial channel structures and mammalian channel models have begun to describe how these sequences lead to analogous high field strength ion binding sites that drive Na+ conduction. Similar complexes have also been identified in unrelated acid sensing ion channels involving glutamate and aspartate side chains that control their selectivity. These studies suggest the possibility of a common origin for Na+ selective binding and transport.

Understanding Sodium Channel Function and Modulation Using Atomistic Simulations of Bacterial Channel Structures.

Sodium channels are chief proteins involved in electrical signaling in the nervous system, enabling critical functions like heartbeat and brain activity. New high-resolution X-ray structures for bacterial sodium channels have created an opportunity to see how these proteins operate at the molecular level. An important challenge to overcome is establishing relationships between the structures and functions of mammalian and bacterial channels. Bacterial sodium channels are known to exhibit the main structural features of their mammalian counterparts, as well as several key functional characteristics, including selective ion conduction, voltage-dependent gating, pore-based inactivation and modulation by local anesthetic, antiarrhythmic and antiepileptic drugs. Simulations have begun to shed light on each of these features in the past few years. Despite deviations in selectivity signatures for bacterial and mammalian channels, simulations have uncovered the nature of the multiion conduction mechanism associated with Na(+) binding to a high-field strength site established by charged glutamate side chains. Simulations demonstrated a surprising level of flexibility of the protein, showing that these side chains are active participants in the permeation process. They have also uncovered changes in protein structure, leading to asymmetrical collapses of the activation gate that have been proposed to correspond to inactivated structures. These observations offer the potential to examine the mechanisms of state-dependent drug activity, focusing on pore-blocking and pore-based slow inactivation in bacterial channels, without the complexities of inactivation on multiple timescales seen in eukaryotic channels. Simulations have provided molecular views of the interactions of drugs, consistent with sites predicted in mammalian channels, as well as a wealth of other sites as potential new drug targets. In this chapter, we survey the new insights into sodium channel function that have emerged from studies of simpler bacterial channels, which provide an excellent learning platform, and promising avenues for mechanistic discovery and pharmacological development.

Selective ion permeation involves complexation with carboxylates and lysine in a model human sodium channel

Bacterial and human voltage-gated sodium channels (Navs) exhibit similar cation selectivity, despite their distinct EEEE and DEKA selectivity filter signature sequences. Recent high-resolution structures for bacterial Navs have allowed us to learn about ion conduction mechanisms in these simpler homo-tetrameric channels, but our understanding of the function of their mammalian counterparts remains limited. To probe these conduction mechanisms, a model of the human Nav1.2 channel has been constructed by grafting residues of its selectivity filter and external vestibular region onto the bacterial NavRh channel with atomic-resolution structure. Multi-μs fully atomistic simulations capture long time-scale ion and protein movements associated with the permeation of Na+ and K+ ions, and their differences. We observe a Na+ ion knock-on conduction mechanism facilitated by low energy multi-carboxylate/multi-Na+ complexes, akin to the bacterial channels. These complexes involve both the DEKA and vestibular EEDD rings, acting to draw multiple Na+ into the selectivity filter and promote permeation. When the DEKA ring lysine is protonated, we observe that its ammonium group is actively participating in Na+ permeation, presuming the role of another ion. It participates in the formation of a stable complex involving carboxylates that collectively bind both Na+ and the Lys ammonium group in a high-field strength site, permitting pass-by translocation of Na+. In contrast, multiple K+ ion complexes with the DEKA and EEDD rings are disfavored by up to 8.3 kcal/mol, with the K+-lysine-carboxylate complex non-existent. As a result, lysine acts as an electrostatic plug that partially blocks the flow of K+ ions, which must instead wait for isomerization of lysine downward to clear the path for K+ passage. These distinct mechanisms give us insight into the nature of ion conduction and selectivity in human Nav channels, while uncovering high field strength carboxylate binding complexes that define the more general phenomenon of Na+-selective ion transport in nature.