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

Abstract

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...

Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac

“…A completely converged average over this degree of freedom cannot be assumed. At other sites, such as in the central pore cavity, anesthetics showed a rather fast rotational tumbling and translational diffusivity in the simulation (11). Such extreme mobility might reduce the cross-relaxation rate, rendering binding undetectable by STD.…”

“…This method depends critically on the availability of a reliable structural model, either from experimental structures or from homology modeling. The success in the strategic placement of 19 F probes based on the predictions of potential isoflurane binding sites from MD simulations (11,12) underscores the power of combining experimental NMR with computational simulations.…”

“…It shares with Na V a similar pharmacological profile in response to volatile anesthetics, such as isoflurane, which inhibits inward currents of NaChBac in a concentrationdependent manner (10). The ease with which NaChBac can be produced in large quantity and high purity makes it a suitable model for high-resolution structural and functional studies of anesthetic binding in voltage-gated ion channels (11,12).…”

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

“…The Na v Bac family lacks the Domain (D) III-DIV linker responsible for fast inactivation (3), enabling molecular-level investigation into this common modulation process. Moreover, key binding regions are well conserved in bacterial and mammalian channels (3,11), and it has been shown that bacterial channels interact with common inhibitory drugs (11)(12)(13)(14). However, questions remain as to how structurally disparate inhibitor molecules can cause qualitatively similar modulation of different mammalian and bacterial Na v channels.…”

Divergent effects of anesthetics on lipid bilayer properties and sodium channel function

“…Another MD flooding simulation and combined electrophysiology study of NaChBac interactions revealed that sevoflurane, at low (0.2 mM) and high (2.0 mM) concentrations, shifted both the voltage-dependence of activation and inactivation toward more hyperpolarized potentials. However, in contrast to the effects of isoflurane, peak Na + current was potentiated at the low sevoflurane concentration, and there was an increase in the rate constant for recovery from inactivation (Barber et al 2014). This acceleration is in contrast to the electrophysiological effects of volatile anesthetics on NaChBac and mammalian Na v , where recovery from inactivation is prolonged (Ouyang et al 2007; Ouyang et al 2009; Herold et al 2014; Purtell et al 2015).…”