Neurotransmitters such as acetylcholine and GABA (gamma-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 GABA(A) 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 alpha1-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.

The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

The firefly luciferase enzyme from Photinus pyralis is probably the best-characterized model system for studying anesthetic-protein interactions. It binds a diverse range of general anesthetics over a large potency range, displays a sensitivity to anesthetics that is very similar to that found in animals, and has an anesthetic sensitivity that can be modulated by one of its substrates (ATP). In this paper we describe the properties of bromoform acting as a general anesthetic (in Rana temporaria tadpoles) and as an inhibitor of the firefly luciferase enzyme at high and low ATP concentrations. In addition, we describe the crystal structure of the low-ATP form of the luciferase enzyme in the presence of bromoform at 2.2-A resolution. These results provide a structural basis for understanding the anesthetic inhibition of the enzyme, as well as an explanation for the ATP modulation of its anesthetic sensitivity.

General anesthetic drugs interact with many receptors in the nervous system, but only a handful of these interactions are critical for producing anesthesia. Over the last 20 years, neuropharmacologists have revealed that one of the most important target sites for general anesthetics is the GABAA receptor. In this review we will discuss what is known about anesthetic – GABAA receptor interactions.

The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-D-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

The biology underlying excessive daytime sleepiness (hypersomnolence) is incompletely understood. After excluding known causes of sleepiness in 32 hypersomnolent patients, we showed that, in the presence of 10 μM γ-aminobutyric acid (GABA), cerebrospinal fluid (CSF) from these subjects stimulated GABA(A) receptor function in vitro by 84.0 ± 40.7% (SD) relative to the 35.8 ± 7.5% (SD) stimulation obtained with CSF from control subjects (Student's t test, t = 6.47, P < 0.0001); CSF alone had no effect on GABA(A) signaling. The bioactive CSF component had a mass of 500 to 3000 daltons and was neutralized by trypsin. Enhancement was greater for α2 subunit- versus α1 subunit-containing GABA(A) receptors and negligible for α4 subunit-containing ones. CSF samples from hypersomnolent patients also modestly enhanced benzodiazepine (BZD)-insensitive GABA(A) receptors and did not competitively displace BZDs from human brain tissue. Flumazenil--a drug that is generally believed to antagonize the sedative-hypnotic actions of BZDs only at the classical BZD-binding domain in GABA(A) receptors and to lack intrinsic activity--nevertheless reversed enhancement of GABA(A) signaling by hypersomnolent CSF in vitro. Furthermore, flumazenil normalized vigilance in seven hypersomnolent patients. We conclude that a naturally occurring substance in CSF augments inhibitory GABA signaling, thus revealing a new pathophysiology associated with excessive daytime sleepiness.

These data are consistent with the idea that the GABA(A) receptor plays a central role in the actions of etomidate. Etomidate exerts its effects on the receptor by binding directly to a specific site or sites on the protein and allosterically enhancing the apparent affinity of GABA for its receptor.

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