James R. Trudell

Coupling of agonist binding to channel gating in the GABAA receptor

Neurotransmitters such as acetylcholine and GABA (γ-aminobutyric acid) mediate rapid synaptic transmission by activating receptors belonging to the gene superfamily of ligand-gated ion channels (LGICs). These channels are pentameric proteins that function as signal transducers, converting chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with a ligand-binding site, triggering a conformational change in the protein that results in the opening of an ion channel. This process, which is known as ‘gating’, occurs rapidly and reversibly, but the molecular rearrangements involved are not well understood. Here we show that optimal gating in the GABAA receptor, a member of the LGIC superfamily, is dependent on electrostatic interactions between the negatively charged Asp 57 and Asp 149 residues in extracellular loops 2 and 7, and the positively charged Lys 279 residue in the transmembrane 2–3 linker region of the α1-subunit. During gating, Asp 149 and Lys 279 seem to move closer to one another, providing a potential mechanism for the coupling of ligand binding to opening of the ion channel.

Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration

Studies using molecular modeling, genetic engineering, neurophysiology/pharmacology, and whole animals have advanced our understanding of where and how inhaled anesthetics act to produce immobility (minimum alveolar anesthetic concentration; MAC) by actions on the spinal cord. Numerous ligand- and voltage-gated channels might plausibly mediate MAC, and specific animo acid sites in certain receptors present likely candidates for mediation. However, in vivo studies to date suggest that several channels or receptors may not be mediators (e.g., &ggr;-aminobutyric acid A, acetylcholine, potassium, 5-hydroxytryptamine-3, opioids, and &agr;2-adrenergic), whereas other receptors/channels (e.g., glycine, N-methyl-d-aspartate, and sodium) remain credible candidates.

A unitary theory of anesthesia based on lateral phase separations in nerve membranes.

This paper relates research on anesthetic effects on lipid membrane systems to mechanisms of neural function. A unitary theory of anesthesia based on anesthetic-induced changes in fluid-solid-phase separations in the lipid region of nerve membranes is presented. It is suggested that anesthetics act by fluidizing nerve membranes to a point where critical lipid regions no longer contain phase separations. As a consequence, the membranes are less able to facilitate the conformational changes in proteins that may be the basis for such membrane events as ion gating, synaptic transmitter release, and transmitter binding to receptors. It is proposed that the anesthetic-modified phase separation behavior of the membrane may alter neural function by a combination of the following effects: inhibition of conformational changes of intrinsic membrane proteins; prevention of the association of protein subunits to form polymeric ion channels; depression of transmitter release by preventing fusion of vesicles containing synaptic transmitter with the membrane of the presynaptic terminal.

Ethanol's molecular targets.

Ethanol produces a wide variety of behavioral and physiological effects in the body, but exactly how it acts to produce these effects is still poorly understood. Although ethanol was long believed to act nonspecifically through the disordering of lipids in cell membranes, proteins are at the core of most current theories of its mechanisms of action. Although ethanol affects various biochemical processes such as neurotransmitter release, enzyme function, and ion channel kinetics, we are only beginning to understand the specific molecular sites to which ethanol molecules bind to produce these myriad effects. For most effects of ethanol characterized thus far, it is unknown whether the protein whose function is being studied actually binds ethanol, or if alcohol is instead binding to another protein that then indirectly affects the functioning of the protein being studied. In this Review, we describe criteria that should be considered when identifying alcohol binding sites and highlight a number of proteins for which there exists considerable molecular-level evidence for distinct ethanol binding sites. For much of the 20th century, it was widely believed that ethanol exerts its effects on neuronal function in a nonspecific manner—perhaps through the disordering of membrane lipids. However, over the past two decades, evidence has mounted that ethanol instead produces its effects by altering the functioning of specific proteins through its interaction with a select few amino acids in those proteins. In this Review with 2 figures and 60 citations, we focus on proteins for which evidence for specific alcohol binding sites has been obtained, and we briefly describe and compare these ethanol receptors.

Methoxyflurane Metabolism and Renal Dysfunction: Clinical Correlation in Man

Serun inorganic fluoride concentration and urinary inorganic fluoride and oxalic acid excretion were found to be markedly elevated in ten patients previously shown to have methoxyflurane-induced renal dysfunction. Five patients with climically evident renal dysfunction had a mean peak serum inorganic fluoride level (190.4 ± 20.9 μ/1) significantly higher (P < 0.02) than that of those with abnormalities in laboratory tests only (105.8 ± 17.0 μ/1). Similarly, patients with clinically evident renal dysfunction had a mean peak oxalic acid excretion (286.8 ± 39.3 mg/24 hours) significantly greater (P < 0.05) than that of those with laboratory abnormalities only (130.6 ± 51.4 mg/24 hours). That patients anesthetized with halothane had insignificant changes in serum inorganic fluoride concentration and oxalic acid excretion indicates that these substances are products of methoxyflurane metabolism. A proposed metabolic pathway to support this hypothesis is presented, as well as evidence to suggest that inorganic fluoride is the substance responsible for methoxyflurane; induced renal dysfunction.

The effect of two inhalation anesthetics of the order of spin-labeled phospholipid vesicles

The order parameter (S′n) of spin-labeled phosphatidylcholine vesicles has been shown to decrease in a concentration-dependent manner with two inhalation anesthetics, halothane and methoxyfluorane. Similar decreases ofS′n are observed in vesicles labeled adjacent to the polar head group and those labeled near the bilayer center. This suggests that inhalation anesthetics cause a generalized fluidization of the membrane rather than a disorder localized in a particular region of the bilayer. Measurements of the isotropic nitrogen hyperfine coupling constant (a′N) show a decrease in polarity of the environment with increasing anesthetic concentrations. The experimental approach of plottingS′n versus anesthetic concentration provides a test of whether anesthetics produce their effects on a per molecule or per volume basis.

Urinary Metabolites of Halothane in Man

The urinary metabolites of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) were investigated in five individuals given trace doses (25 μCi), and in three individuals given large doses (1 mCi) of radioactively labeled 14C-halothane. The latter were donor subjects for heart transplant operations. Separation of the nonvolatile urinary metabolites of halothane was accomplished by chemical extraction, electrophoresis, ion-exchange and high-pressure liquid chromatography, and gas chromatography. Identification of the individual metabolites was by nuclear magnetic resonance and mass spectrometry. Three major metabolites were identified: trifluoro-acetic acid, N-trifluoroacetyl-2-aminoethanol, and N-acetyl-S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine. Smaller unidentified radioactive peaks were also found. The presence of both ethanolamide and cysteine conjugates of halothane is of concern. These urinary products imply the presence of reactive intermediates. The conjugation of such intermediates to proteins and phospholipids may give rise to the high-molecular-weight covalently bound metabolites demonstrated to be present in the liver following halothane anesthesia. Elucidation of the structures of the urinary metabolities provides information important to an understanding of halothane metabolism and its potential hepatotoxicity.

Hypothesis: Inhaled Anesthetics Produce Immobility and Amnesia by Different Mechanisms at Different Sites

Recent evidence supplies new insights regarding the two universal effects of inhaled anesthetics: 1) immobility in response to a noxious stimulus and 2) amnesia. We hypothesize that these two effects result from actions at separate molecular and anatomic sites and that they are produced by different mechanisms. We propose that inhaled anesthetics cause immobility in response to noxious stimuli by an action in the spinal cord at an interface between polar and nonpolar regions. Such a site might be an interfacial region adjacent to membranes or proteins. In contrast, we propose that production of amnesia occurs at a supraspinal site and occurs in a nonpolar environment. An example of such a nonpolar site could be the interior of a phospholipid bilayer or a hydrophobic pocket within a protein.

Minimum alveolar concentrations of noble gases, nitrogen, and sulfur hexafluoride in rats: helium and neon as nonimmobilizers (nonanesthetics)

We assessed the anesthetic properties of helium and neon at hyperbaric pressures by testing their capacity to decrease anesthetic requirement for desflurane using electrical stimulation of the tail as the anesthetic endpoint (i.e., the minimum alveolar anesthetic concentration [MAC]) in rats. Partial pressures of helium or neon near those predicted to produce anesthesia by the Meyer-Overton hypothesis (approximately 80-90 atm), tended to increase desflurane MAC, and these partial pressures of helium and neon produced convulsions when administered alone. In contrast, the noble gases argon, krypton, and xenon were anesthetic with mean MAC values of (+/- SD) of 27.0 +/- 2.6, 7.31 +/- 0.54, and 1.61 +/- 0.17 atm, respectively. Because the lethal partial pressures of nitrogen and sulfur hexafluoride overlapped their anesthetic partial pressures, MAC values were determined for these gases by additivity studies with desflurane. Nitrogen and sulfur hexafluoride MAC values were estimated to be 110 and 14.6 atm, respectively. Of the gases with anesthetic properties, nitrogen deviated the most from the Meyer-Overton hypothesis. Implications: It has been thought that the high pressures of helium and neon that might be needed to produce anesthesia antagonize their anesthetic properties (pressure reversal of anesthesia). We propose an alternative explanation: like other compounds with a low affinity to water, helium and neon are intrinsically without anesthetic effect. (Anesth Analg 1998;87:419-24)

Chronic exposure to anesthetic gases in the operating room.

Halothane present in the ambient atmosphere was continuously measured in each of two operating rooms during the conduct of surgical anesthesia. Concentrations were determined on-line with a mass spectrometer and found to vary with sampling site, breathing system used, and the scavenging system employed to remove overflow anesthetic gases. Concentrations of halothane measured within a 3-foot radius of the anesthesia equipment averaged 8.7 ppm when a nonrebreathing circuit was used (flow 10 l/min), and 4.9 ppm with a semiclosed circle system (flow 4–5 l/min). End-tidal concentrations of halothane averaged 0.21 ppm in 81 samplings from operating room nurses and 0.46 ppm in 36 samplings from anesthetists. Residual concentrations were present in many individuals 16 hours after exposure. A significant reduction in atmospheric contamination of the operating room was obtained by use of appropriate scavenging equipment. The implications of these findings are discussed.