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1.  Volatile anesthetics inhibit sodium channels without altering bulk lipid bilayer properties 
The Journal of General Physiology  2014;144(6):545-560.
Volatile anesthetics act directly on neuronal sodium channels, independently of effects on the lipid bilayer.
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 (Nav). 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 Nav function and lipid bilayer properties. We examined the effects of these agents on Nav 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 Nav 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 Nav 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.
PMCID: PMC4242807  PMID: 25385786
2.  Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function 
ACS Chemical Biology  2014;9(8):1788-1798.
A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.
PMCID: PMC4136704  PMID: 24901212
3.  Isoflurane Reversibly Destabilizes Hippocampal Dendritic Spines by an Actin-Dependent Mechanism 
PLoS ONE  2014;9(7):e102978.
General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However, the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines, a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D, drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus, isoflurane destabilizes spine F-actin, resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus, a locus for anesthetic-induced amnesia, and have important implications for anesthetic effects on synaptic plasticity.
PMCID: PMC4113311  PMID: 25068870
4.  Sodium Channels as Targets for Volatile Anesthetics 
The molecular mechanisms of modern inhaled anesthetics are still poorly understood although they are widely used in clinical settings. Considerable evidence supports effects on membrane proteins including ligand- and voltage-gated ion channels of excitable cells. Na+ channels are crucial to action potential initiation and propagation, and represent potential targets for volatile anesthetic effects on central nervous system depression. Inhibition of presynaptic Na+ channels leads to reduced neurotransmitter release at the synapse and could therefore contribute to the mechanisms by which volatile anesthetics produce their characteristic end points: amnesia, unconsciousness, and immobility. Early studies on crayfish and squid giant axon showed inhibition of Na+ currents by volatile anesthetics at high concentrations. Subsequent studies using native neuronal preparations and heterologous expression systems with various mammalian Na+ channel isoforms implicated inhibition of presynaptic Na+ channels in anesthetic actions at clinical concentrations. Volatile anesthetics reduce peak Na+ current (INa) and shift the voltage of half-maximal steady-state inactivation (h∞) toward more negative potentials, thus stabilizing the fast-inactivated state. Furthermore recovery from fast-inactivation is slowed, together with enhanced use-dependent block during pulse train protocols. These effects can depress presynaptic excitability, depolarization and Ca2+ entry, and ultimately reduce transmitter release. This reduction in transmitter release is more potent for glutamatergic compared to GABAergic terminals. Involvement of Na+ channel inhibition in mediating the immobility caused by volatile anesthetics has been demonstrated in animal studies, in which intrathecal infusion of the Na+ channel blocker tetrodotoxin increases volatile anesthetic potency, whereas infusion of the Na+ channels agonist veratridine reduces anesthetic potency. These studies indicate that inhibition of presynaptic Na+ channels by volatile anesthetics is involved in mediating some of their effects.
PMCID: PMC3316150  PMID: 22479247
sodium channels; volatile anesthetics; presynaptic; anesthetic mechanism
5.  Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery 
The Journal of General Physiology  2011;138(2):249-270.
The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator–activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer–water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs’ partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer–dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.
PMCID: PMC3149818  PMID: 21788612
6.  Isoflurane Inhibits the Tetrodotoxin-resistant Voltagegated Sodium Channel Nav1.8 
Anesthesiology  2009;111(3):591-599.
Voltage-gated sodium channels (Nav) mediate neuronal action potentials. Tetrodotoxin inhibits all Nav isoforms, but Nav1.8 and Nav1.9 are relatively tetrodotoxin-resistant (TTX-r) compared to other isoforms. Nav1.8 is highly expressed in dorsal root ganglion neurons and is functionally linked to nociception, but the sensitivity of TTX-r isoforms to inhaled anesthetics is unclear.
The sensitivities of heterologously expressed rat TTX-r Nav1.8 and endogenous tetrodotoxin-sensitive (TTX-s) Nav to the prototypic inhaled anesthetic isoflurane were tested in mammalian ND7/23 cells using patch-clamp electrophysiology.
From a holding potential of −70 mV, isoflurane (0.53±0.06 mM, ~1.8 MAC at 24°C) reduced normalized peak Na+ current (INa) of Nav1.8 to 0.55±0.03 and of endogenous TTX-s Nav to 0.56±0.06. Isoflurane minimally inhibited INa from a holding potential of −140 mV. Isoflurane did not affect voltage-dependence of activation, but significantly shifted voltage-dependence of steady-state inactivation by −6 mV for Nav1.8 and by −7 mV for TTX-s Nav. IC50 values for inhibition of peak INa were 0.67±0.06 mM for Nav1.8 and 0.66±0.09 mM for TTX-s Nav; significant inhibition occurred at clinically relevant concentrations as low as 0.58 MAC. Isoflurane produced use-dependent block of Nav1.8; at a stimulation frequency of 10 Hz, 0.56±0.08 mM isoflurane reduced INa to 0.64±0.01 vs. 0.78±0.01 for control.
Isoflurane inhibited the tetrodotoxin-resistant isoform Nav1.8 with potency comparable to that for endogenous tetrodotoxin-sensitive Nav isoforms, indicating that sensitivity to inhaled anesthetics is conserved across diverse Nav family members. Block of Nav1.8 in dorsal root ganglion neurons could contribute to the effects of inhaled anesthetics on peripheral nociceptive mechanisms.
PMCID: PMC2756082  PMID: 19672182
7.  Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+ Channel Function 
Anesthesiology  2009;110(3):582-590.
Inhibition of voltage-gated Na+ channels (Nav) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane and desflurane) on Nav1.4, a well characterized pharmacological model for Nav effects.
Na+ currents (INa) from rat Nav1.4 α-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording.
Halogenated inhaled anesthetics reversibly inhibited Nav1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equi-anesthetic concentrations, peak INa was inhibited with a rank order of desflurane > halothane ≈ enflurane > isoflurane ≈ sevoflurane from a physiological holding potential (−80 mV). This suggests that the contribution of Na+ channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (−120 mV), peak INa was inhibited with a rank order of potency for tonic inhibition of peak INa of halothane > isoflurane ≈ sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane.
Five halogenated inhaled anesthetics all inhibit a voltage-gated Na+ channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na+ channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.
PMCID: PMC2699670  PMID: 19225394

Results 1-7 (7)