Ligand-gated Ion Channel Receptors Research Articles

Anesthetic sites on GABAA receptors

Enhancement of GABAA receptor-mediated inhibition is the major candidate mechanism for the molecular action of general anesthetics. The GABAA receptors are members of the cys-loop superfamily of ligand-gated ion channel receptors whose structures are being elucidated. Benzodiazepines, barbiturates and non-barbiturate intravenous anesthetics, ethanol and other alcohols, volatile anesthetics, and neuroactive steroids modulate GABAA receptor function, via allosteric sites on the receptor protein. The benzodiazepine binding sites are located in the extracellular domain at subunit interfaces, similar to agonist binding pockets; these ‘agonist sites’ may be sensitive to other modulators. We showed that modulation by intravenous anesthetics requires residues in the membrane-spanning domain, TM1, while others found that volatile anesthetic modulation requires residues in TM2 and TM3. Affinity labeling of nicotinic acetylcholine receptors with anesthetic ligands by Cohen and associates reveals attachment to these membrane-spanning domains as well as residues in the agonist binding pockets and the pre-TM1 region. We purified bovine brain GABAA receptor protein to homogeneity in mg quantities using refined benzodiazepine affinity chromatography in mild detergent, and photolabeled with [3H]azi-etomidate. A single band on SDS–PAGE corresponding to the GABAA receptor β2/3 subunit was obtained in amounts suitable for microsequencing to identify the binding site.

Anesthetics as Chemical Tools to Study the Structure and Function of Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptor (AChR) is the archetype of the Cys-loop ligand-gated ion channel receptor superfamily. Noncompetitive antagonists inhibit the AChR without interacting directly with agonist sites. Among noncompetitive antagonists, general and local anesthetics have been used for decades to study the structure and function of muscle- as well as neuronal-type AChRs. In this review, we address and update all information regarding the characterization of binding sites and the mechanism of action for n-alkanols, barbiturates, inhalational and dissociative general anesthetics, as well as for tertiary and quaternary local anesthetics. The experimental evidence outlined in this review suggest that: (1) several neuronal-type AChRs might be targets for the pharmacological action of distinct anesthetics; (2) the molecular components of a specific anesthetic locus on a certain receptor type are different from the structural determinants of the site for the same anesthetic on a different receptor type; (3) there are unique binding sites for distinct anesthetics in the same receptor; (4) the affinity of a specific anesthetic depends on the AChR conformational state; (5) anesthetics may inhibit AChRs by different mechanisms including open-channel-blocking, augmenting the desensitization process, and/or inactivating the opening of resting receptors; and (6) some anesthetics may potentiate AChR activity.

AChBP structures for understanding ligand binding in nicotinic receptors

Acetylcholine-binding protein (AChBP) from the mollusc Lymnaea stagnalis is at present the only high-resolution model for the ligand-binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5HT3, GABAA, GABAC and glycine receptors. Here we present crystal structures from remote homologs from other molluscs that will define the variabilities in the binding sites. We will also explore a series of crystal structures of nicotinic receptor agonists and other ligands. These define how cation-pi interactions as well as remote electrostatic compensation contribute to ligand binding in the receptors. These structures also explain the many different data from ligand-binding studies on this pharmaceutically important class of neuronal receptors. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling and drug design.

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon–intron structure as follows: (1) The L2 protein has four transmembrane regions (M1–M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.

Crystal Structure of Acetylcholine-binding Protein from Bulinus truncatus Reveals the Conserved Structural Scaffold and Sites of Variation in Nicotinic Acetylcholine Receptors

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-HT3), gamma-aminobutyric acid (GABA), types A and C, and glycine receptors. Here we present the crystal structure of a remote homolog, AChBP from Bulinus truncatus, which reveals both the conserved structural scaffold and the sites of variation in this receptor family. These include rigid body movements of loops that are close to the transmembrane interface in the receptors and changes in the intermonomer contacts, which alter the pentamer stability drastically. Structural, pharmacological and mutational analysis of both AChBPs shows how 3 amino acid changes in the binding site contribute to a 5-10-fold difference in affinity for nicotinic ligands. Comparison of these structures will be valuable for improving structure-function studies of ligand-gated ion channel receptors, including signal transduction, homology modeling, and drug design.

The Loop C Region of the Murine 5-HT3A Receptor Contributes to the Differential Actions of 5-Hydroxytryptamine and m-Chlorophenylbiguanide

Sequence and predicted structural similarities between members of the Cys loop superfamily of ligand-gated ion channel receptors and the acetylcholine binding protein (AChBP) suggest that the ligand-binding site is formed by six loops that intersect at subunit interfaces. We employed site-directed mutagenesis to investigate the role of amino acids from the loop C region of the murine 5-HT(3AS)R in interacting with two structurally different agonists, serotonin (5-HT) and m-chlorophenylbiguanide (mCPBG). Mutant receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies. Electrophysiological assays were employed to identify changes in response characteristics and relative efficacies of mCPBG and the partial agonist, 2-methyl 5-HT (2-Me5-HT). We have also constructed novel 5-HT and mCPBG docked models of the receptor binding site based on homology models of the AChBP. Both ligand-docked models correlate well with results from mutagenesis and electrophysiological assays. Four key amino acids were identified as being important to ligand binding and/or gating of the receptor. Among these, I228 and D229 are specific for effects mediated by 5-HT compared to mCPBG, indicating a differential interaction of these ligands with loop C. Residues F226 and Y234 are important for both 5-HT and mCPBG interactions. Mutations at F226, I228, and Y234 also altered the relative efficacies of agonists, suggesting a role in the gating mechanism.

P.5.050 The efficacy of treatment with tanakan (EGb 761) in relation to cognitive impairment in patients with cerebrovascular disease

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. The GlyR comprises a pentameric complex that forms a chloride-selective transmembrane channel, which is predominantly expressed in the spinal cord and brain stem. We review the pharmacological and physiological properties of the GlyR and relate this information to more recent insights that have been obtained through the cloning and recombinant expression of the GlyR subunits. We also discuss insights into our understanding of GlyR structure and function that have been obtained by the genetic characterisation of various heritable disorders of glycinergic neurotransmission.

Fishing for allosteric sites on GABA A receptors

GABA A receptors have structural and functional homology with a super-family of cys-loop ligand-gated ion channel receptors including the nicotinic acetylcholine receptors. Amino acid residues involved in ligand-binding pockets are homologous among super-family members, leading to the multiple-loop model of binding sites situated at subunit interfaces, validated by structural studies on the nicotinic acetylcholine receptor and water-soluble snail acetylcholine binding protein. This article will briefly review the literature on the agonist binding sites on the receptor super-family, and then describe the current situation for attempts to identify sites for allosteric modulators on the GABA A receptors. A combination of mutagenesis and photoaffinity labeling with anesthetic ligands has given some leads in this endeavor. Current work by others and ourselves focuses on three putative sites for modulators: (1) within the ion channel domain TM2, near the extracellular end; (2) the agonist binding sites and homologous pockets at other subunit interfaces of the pentameric receptor; and (3) on the linker region stretching from the agonist site loop C to the top of the TM1 region. It is likely that concrete structural information will be forthcoming soon.

Minimal Structural Rearrangement of the Cytoplasmic Pore during Activation of the 5-HT3A Receptor

Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT(3A)) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd2+) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2'C and, to a lesser extent, G-2'C, showed high affinity inhibition with Cd2+ when applied extracellularly in the open state. Cd2+ inhibition in S2'C was attenuated if applied in the presence of an open-channel inhibitor and showed voltage-dependent recovery, indicating a direct effect of Cd2+ in the pore. When applied intracellularly, Cd2+ appeared to bind S2'C receptors in the closed state. The ability of cysteine side chains at the 2' and -2' positions to coordinate Cd2+ in both the native open and closed states of the channel suggests that the cytoplasmic selectivity filter of 5-HT(3A) receptors maintains a narrow pore during channel gating.

Order Changes within Receptor Systems upon Ligand Binding: Receptor Tightening/Oligomerisation and the Interpretation of Binding Parameters

Recent hydrogen–deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (Δ H) and order (Δ S) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of “thermodynamic discrimination” between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the Δ H and Δ S of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.

Positioning of the alpha-subunit isoforms confers a functional signature to gamma-aminobutyric acid type A receptors.

Fast synaptic inhibitory transmission in the CNS is mediated by gamma-aminobutyric acid type A (GABA(A)) receptors. They belong to the ligand-gated ion channel receptor superfamily, and are constituted of five subunits surrounding a chloride channel. Their clinical interest is highlighted by the number of therapeutic drugs that act on them. It is well established that the subunit composition of a receptor subtype determines its pharmacological properties. We have investigated positional effects of two different alpha-subunit isoforms, alpha(1) and alpha(6), in a single pentamer. For this purpose, we used concatenated subunit receptors in which subunit arrangement is predefined. The resulting receptors were expressed in Xenopus oocytes and analyzed by using the two-electrode voltage-clamp technique. Thus, we have characterized gamma(2)beta(2)alpha(1)beta(2)alpha(1), gamma(2)beta(2)alpha(6)beta(2)alpha(6), gamma(2)beta(2)alpha(1)beta(2)alpha(6), and gamma(2)beta(2)alpha(6)beta(2)alpha(1) GABA(A) receptors. We investigated their response to the agonist GABA, to the partial agonist piperidine-4-sulfonic acid, to the noncompetitive inhibitor furosemide and to the positive allosteric modulator diazepam. Each receptor isoform is characterized by a specific set of properties. In this case, subunit positioning provides a functional signature to the receptor. We furthermore show that a single alpha(6)-subunit is sufficient to confer high furosemide sensitivity, and that the diazepam efficacy is determined exclusively by the alpha-subunit neighboring the gamma(2)-subunit. By using this diagnostic tool, it should become possible to determine the subunit arrangement of receptors expressed in vivo that contain alpha(1)- and alpha(6)-subunits. This method may also be applied to the study of other ion channels.

Order Changes within Receptor Systems upon Ligand Binding: Receptor Tightening/Oligomerisation and the Interpretation of Binding Parameters

Recent hydrogen–deuterium exchange experiments have highlighted tightening and loosening of protein structures upon ligand binding, with changes in bonding (ΔH) and order (ΔS) which contribute to the overall thermodynamics of ligand binding. Tightening and loosening show that ligand binding respectively stabilises or destabilises the internal structure of the protein, i.e. it shows positive or negative cooperativity between ligand binding and the receptor structure. In the case of membrane-bound receptors, such as G protein-coupled receptors (GPCRs) and ligand gated ion channel receptors (LGICRs), most binding studies have focussed on association/dissociation constants. Where these have been broken down into enthalpic and entropic contributions, the phenomenon of “thermodynamic discrimination” between antagonists and agonists has often been noted; e.g. for a receptor where agonist binding is predominantly enthalpy driven, antagonist binding is predominantly entropy driven and vice versa. These data have not previously been considered in terms of the tightening, or loosening, of receptor structures that respectively occurs upon positively, or negatively, cooperative binding of ligand. Nor have they been considered in light of the homo- and hetero-oligomerisation of GPCRs and the possibility of ligand-induced changes in oligomerisation. Here, we argue that analysis of the ΔH and ΔS of ligand binding may give useful information on ligand-induced changes in membrane-bound receptor oligomers, relevant to the differing effects of agonists and antagonists.

Comparison of Taurine- and Glycine-induced Conformational Changes in the M2-M3 Domain of the Glycine Receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.

Antipsychotic drugs antagonize human serotonin type 3 receptor currents in a noncompetitive manner.

The serotonin type 3 (5-HT(3)) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). 5-HT(3) receptors play an important role in modulating the inhibitory action of dopamine in mesocorticolimbic brain regions. Neuroleptic drugs are commonly thought to exert their psychopharmacological action mainly through dopamine and serotonin type 2 (5-HT(2)) receptors. Except for clozapine, a direct pharmacological interaction of neuroleptics with 5-HT(3) receptors has not yet been described. Using the concentration-clamp technique, we investigated the effects of flupentixol, various phenothiazines, haloperidol, clozapine and risperidone on Na(+)-inward currents through 5-HT(3) receptors stably expressed in human embryonic kidney 293 cells, and through endogenous 5-HT(3) receptors of murine N1E-115 neuroblastoma cells. In addition, we studied their effects on Ca(2+) influx, measured as a change in intracellular Ca(2+) concentrations ([Ca(2+)](i)). All neuroleptic drugs, but not risperidone, antagonized Na(+)- and Ca(2+)-inward currents evoked by 5-HT (10 microM for 2 s and 1 microM, respectively) in a voltage-independent manner. Only clozapine was a competitive antagonist, while all other compounds turned out to be noncompetitive. Fluphenazine and haloperidol affected membrane anisotropy at concentrations below their IC(50) values, indicating that a change in membrane anisotropy might contribute to their antagonistic effect at the 5-HT(3) receptor. Only structure analogues of flupentixol and fluphenazine with a lipophilic side chain were potent antagonists against 5-HT-evoked Na(+) and Ca(2+) currents. Since 5-HT(3) receptors modulate mesolimbic and mesocortical dopaminergic activity, the functional antagonism of neuroleptics at 5-HT(3) receptors may contribute to their antipsychotic efficacy and may constitute a not yet recognized pharmacological principle of these drugs.

In vitro inhibition of recombinant ligand-gated ion channels by high concentrations of milnacipran

Tricyclic antidepressants are known to inhibit various ligand-gated ion-channel (LGIC) receptors, and some of their clinical features may be associated with this activity. The effects of milnacipran, a selective inhibitor of the reuptake of serotonin and noradrenaline, on LGIC receptors have not yet been investigated on such ion-channel receptors. To determine the in vitro effect of milnacipran on four recombinant LGIC receptors, nicotinic acetylcholine, N-methyl-D-aspartate, gamma-amino butyric acid (GABA) and 5-hydroxytryptamine3A receptors. Receptors were expressed in Xenopus oocytes and LGIC activity measured using a two-voltage clamp technique. There was no interaction at the GABA receptor. The results at the other LGIC receptors showed that they could be inhibited by high concentrations of milnacipran. At high concentrations, milnacipran can inhibit certain LGICs. It is, however, unlikely that these interactions have any clinical consequence under normal therapeutic conditions, since the concentrations required are considerably higher than those achieved in plasma of treated patients.

Receptor binding thermodynamics at the neuronal nicotinic receptor.

Simple determination of K(A) or K(D) values makes it possible to calculate the standard free energy DeltaG degrees = -RTlnK(A) = RT lnK(D)(T= 298.15 K) of the binding equilibrium but not that of its two components as defined by the Gibbs equation DeltaG degrees = DeltaH degrees - TDeltaS degrees where DeltaH degrees and DeltaS degrees are the equilibrium standard enthalpy and entropy, respectively. Recently, it has been shown that the relative DeltaH degrees and DeltaS degrees magnitudes can often give a simple "in vitro" way for discriminating "the effect", that is the manner in which the drug interferes with the signal transduction pathways. This particular effect, called "thermodynamic discrimination", results from the fact that binding of antagonists may be enthalpy-driven and that of agonists entropy-driven, or vice-versa. In the past, the thermodynamic discrimination was reported for the beta-adrenergic G-protein-coupled receptor (GPCR) and confirmed later for adenosine A(1), A(2A) and A(3) receptors. Moreover, it has been found that the binding of all ligand-gated ion-channel receptors (LGICR) investigated was thermodynamically discriminated. In particular, affinity constants for typical neuronal nicotinic receptor ligands were obtained by both saturation and inhibition experiments with the radioligand [(3)H]-cytisine, a ganglionic nicotinic agonist. Thermodynamic parameters indicated that agonistic binding was both enthalpy- and entropy-driven, while antagonistic binding was totally entropy-driven. These results have shown that neuronal nicotinic receptor agonists and antagonists were thermodynamically discriminated. On these grounds, the thermodynamic behaviour makes it possible to discriminate drug pharmacological profiles in vivo through binding experiments in vitro.

Role of Charged Residues in Coupling Ligand Binding and Channel Activation in the Extracellular Domain of the Glycine Receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.

A vertical flow chamber for Xenopus oocyte electrophysiology and automated drug screening

Xenopus laevis oocytes are used extensively in the study of ion channel coupled receptors. Efficient use of oocytes for ion channel characterization requires a system that is inherently stable, reproducible, minimizes drug volumes, and maximizes oocyte longevity. We have constructed a vertical flow oocyte recording chamber to address the aforesaid issues, where the oocyte is placed in a funnel-shaped chamber and perfused from the bottom of the funnel. The vertical rather than horizontal flow of perfusate results in an unusually stable environment for oocyte recording. Two-electrode voltage clamp recordings from a single oocyte are acquired easily and routinely over several hours while maintaining stable baseline currents and reproducible response profiles. Chamber characteristics were tested using a serotonin ligand-gated ion channel receptor (5-HT 3R). Data obtained from this system corresponds well with published data. To further test the stability and reliability of this perfusion chamber, we constructed an automated oocyte perfusion system utilizing a commonly available HPLC autosampler. We were able to obtain dose–response curves for various 5-HT 3AR ligands using the automated perfusion system with minimal user intervention. Such a system can easily satisfy need for automated oocyte electrophysiology in academic settings, especially small to medium sized laboratories.

Cannabinoid CB1 receptor and serotonin 3 receptor subunit A (5-HT3A) are co-expressed in GABA neurons in the rat telencephalon.

Among all described serotonin (5-HT) receptors in mammals, the type three (5-HT3) is the only ligand-gated ion channel receptor for serotonin. By using double in situ hybridization histochemistry, we found co-expression of the functional 5-HT3A subunit of the 5-HT3 receptor and the central CB1 cannabinoid receptor in neurons of the rat telencephalon. Double-labeled 5-HT3A/CB1 neurons were found in the anterior olfactory nucleus, superficial and deep layers of the cortex, hippocampal formation (hippocampus, dentate gyrus, subiculum, and entorhinal cortex) and amygdala. Analysis of the proportion of neurons co-expressing 5-HT3A and CB1 receptors in the cortex and amygdala showed that, depending on the brain region, 37-53% of all neurons expressing the 5-HT3A subunit also expressed CB1 transcripts; 16-72% of the total population of neurons expressing CB1 mRNA co-expressed the 5-HT3A subunit. By using a combination of double in situ hybridization and immunohistochemistry, we demonstrated that 5-HT3A/CB1-expressing neurons contained the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). These results imply that in distinct regions of the telencephalon, GABA neurons that react to cannabinoids may also be responsive to serotonin through 5-HT3 receptors. Cellular coexistence of 5-HT3A and CB1 transcripts in interneurons of the cortex, hippocampal formation, and amygdala suggest possible interactions between the cannabinoid and serotonergic systems at the level of GABA neurotransmission in brain areas involved in cognition, memory, and emotion.

Does Serotonin Play a Role in Epilepsy?

Studies in experimental models have suggested a potential role for serotonergic transmission in epilepsy, and interest in this research has been increased by the development of positron emission tomography (PET) ligands that can be used to study 5-hydroxytryptamine (5-HT) receptors and transporters. The serotonergic system is very complex. At least 13 distinct G protein-coupled 5-HT receptors and one ligand-gated ion channel receptor (5-HT(3)) are divided into seven distinct classes (5-HT(1) to 5-HT(7)) ((1)). The receptors vary widely in their distribution and effects, innervating vascular structures and gut smooth muscle as well as neuronal tissue. Several receptor subtypes may be relevant to epilepsy.