Comparative Study of the Gating Motif and C-type Inactivation in Prokaryotic Voltage-gated Sodium Channels

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...

Section: Significancementioning

confidence: 99%

“…Although crystal structures are not yet reported, the bacterial Na + channel NaChBac has been extensively characterized by electrophysiology (22,(28)(29)(30)(31)(32)(33)(34)(35)(36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37).…”

mentioning

confidence: 99%

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

Barber

Carnevale

Klein

et al. 2014

“…The sodium channel from Bacillus halodurans (NaChBac), the first bacterial sodium channel to be identified, has been well-characterized with respect to its conductance properties (4). Since then a large number of other members of the prokaryotic voltage-gated sodium channel family have been identified and shown to be close sequence and structural homologs (5,6). Several crystal structures of bacterial sodium channels have been published recently (7)(8)(9)(10); in these, the proteins are present in different functional states.…”

mentioning

confidence: 99%

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel

Ulmschneider

Bagnéris

McCusker

et al. 2013

161

248

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp.(NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multiion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate. V oltage-gated cation channels are proteins that produce electrical signals in neurons and other excitable cells to regulate muscle contraction, gene expression, and release of hormones and neurotransmitters among other functions. In response to a change in transmembrane electrical potential, the channels open pores through which ions move passively across the membrane. The large family of cation channels includes those selective for sodium, potassium, or calcium. The opening and closing of these ion-specific channels is carefully choreographed to produce the electrical signals required by the nervous system for rapid signal transduction (1).Voltage-gated sodium channels have been causally linked with a wide range of neurological and cardiovascular diseases and hence are important pharmaceutical drug-development targets (2, 3). Eukaryotic voltage-gated sodium channels are large, singlechain polypeptides, consisting of 24 transmembrane (TM) helices that form four homologous repeats, each contributing both a voltage sensor and a pore domain; the latter are arranged to form a central Na + -selective transmembrane pathway. Bacterial voltagegated sodium channels are far simpler, consisting of four polypeptide chains, each of which is composed of six TM segments, with segments TM1-TM4 forming the voltage sensors and TM5-TM6 forming the pore domains. The TM5-TM6 segment...

“…However, in 2001 Ren et al (13) isolated NaChBac, a simple prokaryotic sodium channel from Bacillus halodurans that, like potassium channels, but unlike eukaryotic sodium channels, is composed of a single 6TM domain. Subsequently, many more close homologues from both gram-positive and gram-negative sources have been identified (14)(15)(16). In NaChBac, it has been shown that, as predicted, four monomers assemble to form an active channel (17).…”

mentioning

confidence: 99%

Synchrotron radiation circular dichroism spectroscopy-defined structure of the C-terminal domain of NaChBac and its role in channel assembly

Powl

O’Reilly

Miles

et al. 2010

Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining highresolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na V ) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an α-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of ∼20 α-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na V sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.membrane protein assembly | membrane protein structure | synchrotron radiation circular dichroism (SRCD) spectroscopy | voltage-gated sodium channel | secondary structure S odium, calcium, and potassium channels form a family of integral membrane proteins that selectively regulate ion conductance across an otherwise impermeable lipid membrane. In eukaryotic voltage-gated sodium and calcium channels the functional unit is formed by assembly of four homologous domains of a single polypeptide chain. Each domain is composed of six transmembrane (TM) helices, with the four N-terminal helices forming the voltage-sensing subdomain and the two C-terminal helices comprising the pore subdomain. In contrast, in both eukaryotic and prokaryotic potassium channels the functional unit is a homo-tetramer formed from identical monomers that each correspond to one of the sodium or calcium channel domains (1). The crystal structures of a number of potassium channels have been determined (2-4), which have confirmed the tetrameric nature of the channels and details of their transmembrane (TM) regions. The structures of the extramembranous C-terminal domains of most of these channels, however, were not defined in the crystal structures, either because they had been removed after assembly (KcsA) (2) in order to improve crystallization, or because they were disordered in the crystal (KvAP and MlotiK1) (3, 4).The extramembranous termini of potassium channels appear to play important roles in channel assembly, primarily as tetramerization domains. Examples include the N-terminal T1 domain of Shaker channels (5) and C-terminal do...