Marco Pistis

The 5-HT3B subunit is a major determinant of serotonin-receptor function

The neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) mediates rapid excitatory responses through ligand-gated channels (5-HT3 receptors). Recombinant expression of the only identified receptor subunit (5-HT3A) yields functional 5-HT3 receptors. However, the conductance of these homomeric receptors (sub-picosiemens) is too small to be resolved directly, and contrasts with a robust channel conductance displayed by neuronal 5-HT3 receptors (9–17 pS). Neuronal 5-HT3 receptors also display a permeability to calcium ions and a current–voltage relationship that differ from those of homomeric receptors,. Here we describe a new class of 5-HT3-receptor subunit (5-HT3B). Transcripts of this subunit are co-expressed with the 5-HT3A subunit in the amygdala, caudate and hippocampus. Heteromeric assemblies of 5-HT3A and 5-HT3B subunits display a large single-channel conductance (16 pS), low permeability to calcium ions, and a current–voltage relationship which resembles that of characterized neuronal 5-HT3 channels. The heteromeric receptors also display distinctive pharmacological properties. Surprisingly, the M2 region of the 5-HT3B subunit lacks any of the structural features that are known to promote the conductance of related receptors. In addition to providing a new target for therapeutic agents, the 5-HT3B subunit will be a valuable resource for defining the molecular mechanisms of ion-channel function.

General anaesthetic action at transmitter-gated inhibitory amino acid receptors

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.

The interaction of general anaesthetics and neurosteroids with GABAA and glycine receptors

The positive allosteric effects of four structurally distinct general anaesthetics (propofol, pentobarbitone, etomidate and 5alpha-pregnan-3alpha-ol-20-one [5alpha3alpha]) upon recombinant GABA(A) (alpha6beta3gamma2L), invertebrate GABA (RDL) and glycine (alpha1) receptors expressed in Xenopus laevis oocytes have been determined. Propofol and pentobarbitone enhanced agonist (GABA or glycine as appropriate) evoked currents at GABA(A), glycine, and RDL receptors, whereas etomidate and 5alpha3alpha were highly selective for the GABA(A) receptor. Utilizing site-directed mutagenesis, we demonstrate that the nature of the interaction of propofol, pentobarbitone and etomidate (but not 5alpha3alpha) with mammalian and invertebrate ionotropic GABA receptors depends critically upon the nature of a single amino acid located in the second transmembrane region (TM2) of these receptors. These data are discussed in relation to the specificity of action of general anaesthetics.

Complementary regulation of anaesthetic activation of human (α6β3γ2L) and Drosophila (RDL) GABA receptors by a single amino acid residue

1 The influence of a transmembrane (TM2) amino acid located at a homologous position in human β1 (S290) and β3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2 GABA‐evoked currents mediated by human wild‐type (WT) α6β3γ2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated α6β3γ2L, but not RDL, receptors. 3 GABA‐evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. α6β3N289Mγ2L) were potentiated by propofol or pentobarbitone with ≈2‐fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4 Incorporation of the homologous GABAAβ1‐subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous β3‐subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5 The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (α6β3γ2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.

The Selective Interaction of Neurosteroids with the GABAA Receptor

Some endogenous pregnane steroids have long been known to produce rapid sedative and anesthetic effects (1). The speed of onset of these behavioral effects precludes a genomic mechanism of action for such steroids, but it was not until Harrison and Simmonds (2)demonstrated that a synthetic steroidal anesthetic, alphaxalone (3α-hydroxy-5α-pregnane-11,20-dione), selectively enhanced the interaction of GABA with the GABAA receptor, that a logical mechanism to explain the behavioral effects of these compounds emerged. GABA acting via the GABAA receptor mediates much of the “fast” inhibitory synaptic transmission in the mammalian brain (3).The GABAA receptor is a member of the cysteine-cysteine loop transmitter-gated ion channel family that includes glycine, nicotinic, and 5-HT3 receptors (4).Upon activation by GABA, the associated chloride selective ion channel is opened that increases neuronal membrane conductance and effectively shunts the influence of excitatory transmitters such as glutamate (3).

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

The γ‐aminobutyric acid (GABA)‐modulatory and GABA‐mimetic actions of etomidate at mammalian GABAA receptors are favoured by β2‐ or β3‐ versus β1‐subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β2 N290, β3 N289, β1 S290). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two‐point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate‐insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. Complementary RNA‐injected oocytes expressing the wild type Rdl GABA receptor and voltage‐clamped at −60 mV responded to bath applied GABA with a concentration‐dependent inward current response and a calculated EC50 for GABA of 20±0.4 μM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (RdlM314N) or a serine (RdlM314S) also exhibited a concentration‐dependent inward current response to GABA, but in both cases with a reduced EC50 of 4.8±0.2 μM. Utilizing the appropriate GABA EC10, etomidate (300 μM) had little effect on the agonist‐evoked current of the wild type Rdl receptor. By contrast, at RdlM314N receptors, etomidate produced a clear concentration‐dependent enhancement of GABA‐evoked currents with a calculated EC50 of 64±3 μM and an Emax of 68±2% (of the maximum response to GABA). The actions of etomidate at RdlM314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μMR‐(+)‐etomidate and S‐(−)‐etomidate enhancing the current induced by GABA (EC10) to 52±6% and 12±1% of the GABA maximum respectively. The effects of this mutation were selective for etomidate as the GABA‐modulatory actions of 1 mM pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and RdlM314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one (Rdl=25±4% of the GABA maximum) was not altered by this mutation (RdlM314N=18±3% of the GABA maximum). Etomidate acting at β1 (S290)‐containing mammalian GABAA receptors is known to produce only a modest GABA‐modulatory effect. Similarly, etomidate acting at RdlM314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to RdlM314N receptors. Etomidate acting at human α6β3γ2L receptors is known to produce a large enhancement of GABA‐evoked currents and at higher concentrations this anaesthetic directly activates the GABAA receptor complex. Mutation of the human β3 subunit asparagine to methionine (β3 N289M found in the equivalent position in Rdl completely inhibited both the GABA‐modulatory and GABA‐mimetic action of etomidate (10–300 μM) acting at α6β3 N289Mγ2L receptors. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

The Interaction of Intravenous Anesthetic Agents with Native and Recombinant GABAA Receptors

The γ-aminobutyric acid type-A (GABAA) receptor is a ligand-gated, anion-selective, ion channel that exists as a pentameric complex of structurally homologous subunits (Sieghart, 1995; Smith and Olsen, 1995). Four families of subunit, termed α, β, δ, and γ, whose members may co-assemble to create GABAA receptors with differential biophysical and pharmacological properties, are currently recognized (Burt and Kamatchi, 1991; Macdonald and Angelotti, 1993; Whiting et al., 1995). GABAA receptor isoforms mediate the majority of the inhibitory action of GABA within the central nervous system (CNS), the activation of postsynaptically located GABAA receptors resulting in an increase in membrane conductance, predominantly to chloride ions, which shunts the influence of excitatory neurotransmitters, such as glutamate (Mody et al., 1994).

Endocannabinoid Signaling in Motivation, Reward, and Addiction

Evidence suggests that the endocannabinoid system has been conserved in the animal kingdom for 500 million years, and this system influences many critical behavioral processes including associative learning, reward signaling, goal-directed behavior, motor skill learning, and action-habit transformation. Additionally, the neurotransmitter dopamine has long been recognized to play a critical role in the processing of natural rewards, as well as of motivation that regulates approach and avoidance behavior. This motivational role of dopamine neurons is also based upon the evidence provided by several studies investigating disorders of dopamine pathways such as drug addiction and Parkinsons disease. From an evolutionary point of view, individuals engage in behaviors aimed at maximizing and minimizing positive and aversive consequences, respectively. Accordingly, those with the greatest fitness have a better potential to survival. Hence, deviations from fitness can be viewed as a part of the evolutionary process by means of natural selection. Given the long evolutionary history of both the endocannabinoid and dopaminergic systems, it is plausible that they must serve as fundamental and basic modulators of physiological functions and needs. Notably, endocannabinoids regulate dopamine neuronal activity and its influence on behavioral output. The goal of this chapter is to examine the endocannabinoid influence on dopamine signaling specifically related to (i) those behavioral processes that allow us to successfully adapt to ever-changing environments (i.e., reward signaling and motivational processes) and (ii) derangements from behavioral flexibility that underpin drug addiction.