Distinctly different interactions of anesthetic and nonimmobilizer with transmembrane channel peptides.

Although it plays no clinical role in general anesthesia, gramicidin A, a transmembrane channel peptide, provides an excellent model for studying the specific interaction between volatile anesthetics and membrane proteins at the molecular level. We show here that a pair of structurally similar volatile anesthetic and nonimmobilizer (nonanesthetic), 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1, 2-dichlorohexafluorocyclobutane (F6), respectively, interacts differently with the transmembrane peptide. With 400 microM gramicidin A in a vesicle suspension of 60 mM phosphatidylcholine-phosphatidylglycerol (PC/PG), the intermolecular cross-relaxation rate constants between (19)F of F3 and (1)H in the chemical shift regions for the indole and backbone amide protons were 0.0106 +/- 0.0007 (n = 12) and 0.0105 +/- 0.0014 (n = 8) s(-1), respectively. No cross-relaxation was measurable between (19)F of F6 and protons in these regions. Sodium transport study showed that with 75 microM gramicidin A in a vesicle suspension of 66 mM PC/PG, F3 increased the (23)Na apparent efflux rate constant from 149.7 +/- 7.2 of control (n = 3) to 191.7 +/- 12.2 s(-1) (n = 3), and the apparent influx rate constant from 182.1 +/- 15.4 to 222.8 +/- 21.7 s(-1) (n = 3). In contrast, F6 had no effects on either influx or efflux rate. It is concluded that the ability of general anesthetics to interact with amphipathic residues near the peptide-lipid-water interface and the inability of nonimmobilizer to do the same may represent some characteristics of anesthetic-protein interaction that are of importance to general anesthesia.     

Isoflurane alters the structure and dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.     

Molecular mapping of general anesthetic sites in a voltage-gated ion channel

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.     

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.     

Effects of volatile anesthetic on channel structure of gramicidin A

Volatile anesthetic agent, 1-chloro-1,2,2-trifluorocyclobutane (F3), was found to alter gramicidin A channel function by enhancing Na(+) transport (. Biophys. J. 77:739-746). Whether this functional change is associated with structural alternation is evaluated by circular dichroism and nuclear magnetic resonance spectroscopy. The circular dichroism and nuclear magnetic resonance results indicate that at low millimolar concentrations, 1-chloro-1,2,2-trifluorocyclobutane causes minimal changes in gramicidin A channel structure in sodium dodecyl sulfate micelles. All hydrogen bonds between channel backbones are well maintained in the presence of 1-chloro-1,2,2-trifluorocyclobutane, and the channel structure is stable. The finding supports the notion that low affinity drugs such as volatile anesthetics and alcohols can cause significant changes in protein function without necessarily producing associated changes in protein structure. To understand the molecular mechanism of general anesthesia, it is important to recognize that in addition to structural changes, other protein properties, including dynamic characteristics of channel motions, may also be of functional significance.     

Different distribution of fluorinated anesthetics and nonanesthetics in model membrane: a 19F NMR study.

Despite their structural resemblance, a pair of cyclic halogenated compounds, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), exhibit completely different anesthetic properties. Whereas the former is a potent general anesthetic, the latter produces no anesthesia. Two linear compounds, isoflurane and 2,3-dichlorooctofluorobutane (F8), although not a structural pair, also show the same anesthetic discrepancy. Using 19F nuclear magnetic spectroscopy, we investigated the time-averaged submolecular distribution of these compounds in a vesicle suspension of phosphatidylcholine lipids. A two-site exchange model was used to interpret the observed changes in resonance frequencies as a function of the solubilization of these compounds in membrane and in water. At clinically relevant concentrations, the anesthetics F3 and isoflurane distributed preferentially to regions of the membrane that permit easy contact with water. The frequency changes of these two anesthetics can be well characterized by the two-site exchange model. In contrast, the nonanesthetics F6 and F8 solubilized deeply into the lipid core, and their frequency change significantly deviated from the prediction of the model. It is concluded that although anesthetics and nonanesthetics may show similar hydrophobicity in bulk solvents such as olive oil, their distributions in various regions in biomembranes, and hence their effective concentrations at different submolecular sites, may differ significantly.     

Volatile anesthetics inhibit sodium channels without altering bulk lipid bilayer properties

Although general anesthetics are clinically important and widely used, their molecular mechanisms of action remain poorly understood. Volatile anesthetics such as isoflurane (ISO) are thought to alter neuronal function by depressing excitatory and facilitating inhibitory neurotransmission through direct interactions with specific protein targets, including voltage-gated sodium channels (Na_v). Many anesthetics alter lipid bilayer properties, suggesting that ion channel function might also be altered indirectly through effects on the lipid bilayer. We compared the effects of ISO and of a series of fluorobenzene (FB) model volatile anesthetics on Na_v function and lipid bilayer properties. We examined the effects of these agents on Na_v in neuronal cells using whole-cell electrophysiology, and on lipid bilayer properties using a gramicidin-based fluorescence assay, which is a functional assay for detecting changes in lipid bilayer properties sensed by a bilayer-spanning ion channel. At clinically relevant concentrations (defined by the minimum alveolar concentration), both the FBs and ISO produced prepulse-dependent inhibition of Na_v and shifted the voltage dependence of inactivation toward more hyperpolarized potentials without affecting lipid bilayer properties, as sensed by gramicidin channels. Only at supra-anesthetic (toxic) concentrations did ISO alter lipid bilayer properties. These results suggest that clinically relevant concentrations of volatile anesthetics alter Na_v function through direct interactions with the channel protein with little, if any, contribution from changes in bulk lipid bilayer properties. Our findings further suggest that changes in lipid bilayer properties are not involved in clinical anesthesia.     

Are neuronal voltage-gated calcium channels valid cellular targets for general anesthetics?

The effects of anesthetics and analgesics on ion channels have been the subject of intense research since recent reports of direct actions of anesthetic molecules on ion channel proteins. It is now known that ligand-gated channels, particularly γ-amino-butyric acid (GABA_A) and N-methyl-D-aspartate (NMDA) receptors, play a key role in mediating anesthetic actions, but these channels are unable to account for all aspects of clinical anesthesia such as loss of consciousness, immobility, analgesia, amnesia and muscle relaxation. Furthermore, an assortment of voltage-gated and background channels also display anesthetic sensitivity and a key question arises: What role do these other channels play in clinical anesthesia? These channels have overlapping physiological roles and pharmacological profiles, making it difficult to assign aspects of the anesthetic state to individual channel types. Here, we will focus on the function of neuronal voltage-gated calcium channels in mediating the effects of general anesthetics.Key words: nitrous oxide, low-threshold calcium channel, nociception     

Molecular dynamics simulations of C2F6 effects on gramicidin A: implications of the mechanisms of general anesthesia

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.     

General anesthetic binding to gramicidin A: the structural requirements

There is a distinct possibility that general anesthetics exert their action on the postsynaptic receptor channels. The structural requirements for anesthetic binding in transmembrane channels, however, are largely unknown. High-resolution (1)H nuclear magnetic resonance and direct photoaffinity labeling were used in this study to characterize the volatile anesthetic binding sites in gramicidin A (gA) incorporated into sodium dodecyl sulfate (SDS) micelles and into dimyristoylphosphatidylcholine (DMPC) bilayers, respectively. To confirm that the structural arrangement of the peptide side chains can affect anesthetic binding, gA in nonchannel forms in methanol was also analyzed. The addition of volatile anesthetic halothane to gA in SDS with a channel conformation caused a concentration-dependent change in resonant frequencies of the indole amide protons of W9, W11, W13, and W15, with the most profound changes in W9. These frequency changes were observed only for gA carefully prepared to ensure a channel conformation and were absent for gA in methanol. For gA in DMPC bilayers, direct [(14)C]halothane photolabeling and microsequencing demonstrated dominant labeling of W9, less labeling of W11 and W13, and no significant labeling of W15. In methanol, gA showed much less labeling of any residues. Inspection of the 3-D structure of gA suggests that the spatial arrangements of the tryptophan residues in the channel form of gA, combined with the amphiphilic regions of lipid, create a favorable anesthetic binding motif.     

The gramicidin ion channel: a model membrane protein

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.     

The gramicidin ion channel: A model membrane protein

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.     

Membrane structural perturbations caused by anesthetics and nonimmobilizers: a molecular dynamics investigation.

The structural perturbations of the fully hydrated dimyristoyl-phosphatidylcholine bilayer induced by the presence of hexafluoroethane C(2F6), a "nonimmobilizer," have been examined by molecular dynamics simulations and compared with the effects produced by halothane CF3CHBrCl, an "anesthetic," on a similar bilayer (DPPC) (Koubi et al., Biophys. J. 2000. 78:800). We find that the overall structure of the lipid bilayer and the zwitterionic head-group dipole orientation undergo only a slight modification compared with the pure lipid bilayer, with virtually no change in the potential across the interface. This is in contrast to the anesthetic case in which the presence of the molecule led to a large perturbation of the electrostatic potential across to the membrane interface. Similarly, the analysis of the structural and dynamical properties of the lipid core are unchanged in the presence of the nonimmobilizer although there is a substantial increase in the microscopic viscosity for the system containing the anesthetic. These contrasting perturbations of the lipid membrane caused by those quite similarly sized molecules may explain the difference in their physiological effects as anesthetics and nonimmobilizers, respectively.     

Exploring Volatile General Anesthetic Binding to a Closed Membrane-Bound Bacterial Voltage-Gated Sodium Channel via Computation

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics “flooding” simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the “fenestrations” present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.     

Study,by use of coarse-grained models,of the functionally crucial residues and allosteric pathway of anesthetic regulation of the Gloeobacter violaceus ligand-gated ion channel

Although pentameric ligand-gated ion channels (pLGICs) have been found to be the targets of general anesthetics, the mechanism of the effects of anesthetics on pLGICs remains elusive. pLGICs from Gloeobacter violaceus (GLIC) can be inhibited by the anesthetic ketamine. X-ray crystallography has shown that the ketamine binding site is distant from the channel gate of the GLIC. It is still not clear how ketamine controls the function of the GLIC by long-range allosteric regulation. In this work, the functionally crucial residues and allosteric pathway of anesthetic regulation of the GLIC were identified by use of a coarse-grained thermodynamic method developed by our group. In our method, the functionally crucial sites were identified as the residues thermodynamically coupled with binding of ketamine. The results from calculation were highly consistent with experimental data. Our study aids understanding of the mechanism of the anesthetic action of ketamine on the GLIC by long-range allosteric modulation.     

The effect of anesthetics on hydrogen bonds An infrared study at low anesthetic concentrations

It is shown that a striking parallelism exists between the anesthetic potency of general halocarbon anesthetics and their influence on the hydrogen bond association constants in N-H…OC type hydrogen bonds, important for shaping the ion channels. It is further shown that the effect of potent anesthetics (which contain an acidic hydrogen) on the free/associated ratio in such hydrogen bonds is still significant at clinical anesthetic concentrations. It is argued that the results are in keeping with a pluralistic theory of anesthesia based on both hydrophobic and polar interactions.     

Liquid General Anesthetics Lower Critical Temperatures in Plasma Membrane Vesicles

A large and diverse array of small hydrophobic molecules induce general anesthesia. Their efficacy as anesthetics has been shown to correlate both with their affinity for a hydrophobic environment and with their potency in inhibiting certain ligand-gated ion channels. In this study we explore the effects that n-alcohols and other liquid anesthetics have on the two-dimensional miscibility critical point observed in cell-derived giant plasma membrane vesicles (GPMVs). We show that anesthetics depress the critical temperature (T_c) of these GPMVs without strongly altering the ratio of the two liquid phases found below T_c. The magnitude of this affect is consistent across n-alcohols when their concentration is rescaled by the median anesthetic concentration (AC₅₀) for tadpole anesthesia, but not when plotted against the overall concentration in solution. At AC₅₀ we see a 4°C downward shift in T_c, much larger than is typically seen in the main chain transition at these anesthetic concentrations. GPMV miscibility critical temperatures are also lowered to a similar extent by propofol, phenylethanol, and isopropanol when added at anesthetic concentrations, but not by tetradecanol or 2,6 diterbutylphenol, two structural analogs of general anesthetics that are hydrophobic but have no anesthetic potency. We propose that liquid general anesthetics provide an experimental tool for lowering critical temperatures in plasma membranes of intact cells, which we predict will reduce lipid-mediated heterogeneity in a way that is complimentary to increasing or decreasing cholesterol. Also, several possible implications of our results are discussed in the context of current models of anesthetic action on ligand-gated ion channels.     

Volatile anesthetics inhibit the ion flux through Ca2+-activated K+ channels of rat glioma C6 cells

Ca2+-activated K+ channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes. The ion flux through the channels was studied with 86Rb+ after addition of a Ca2+-ionophore to the incubation medium. Both the influx and efflux of 86Rb+ through these Ca2+-activated K+ channels were inhibited by the general anesthetic halothane (at clinical concentrations). Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca2+-activated K+ channels at clinical concentrations. Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.     

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA_A/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.