Ligand-gated Ion Channel Receptor Superfamily Research Articles

Effects of RIC-3 on the expression profile of serotonin type 3A receptors.

Serotonin type 3A (5-HT3A) receptors belong to the Cys-loop ligand-gated ion channel receptor superfamily. 5-HT3A receptors are found in high densities in certain brain regions, have an involvement with brain-gut signaling circuitry and other non-serotonergic synaptic activities, which make them effective and potential therapeutic targets for treatments of many conditions such as irritable bowel syndrome, chemotherapy-induced vomiting, inflammation, and psychiatric disorders. RIC-3, a transmembrane protein and endoplasmic reticulum resident chaperone, acts as a molecular chaperone of cation-conducting Cys-loop receptors, both serotonin type 3A (5-HT3A) and nicotinic acetylcholine receptors. RIC-3 is needed for efficient receptor folding, assembly and trafficking/functional surface expression. RIC-3 interacts directly with the intracellular domain of serotonin type 3A subunits (ICD). RIC-3 has two binding sites with a shared duplicated motif in 5HT3A subunits, one in the MX-helix and one in the MAM4-helix. Toward drug discovery based on protein-protein interactions, we here aimed to determine the effect of RIC-3 on the expression profile of serotonin type 3A receptors. For this study, 5-HT3A constructs were genetically engineered, and the functional surface expression was examined using two-electrode voltage clamp and immunoblot analysis/western blot. We found that when 5-HT3A channels were co-injected with RIC-3, RIC-3 inhibits 5-HT3A current amplitudes elicited by serotonin in Xenopus oocytes; Ala replacements at residues W347, R349, and L353 (MX) or residues W447, R449, and L454 (MAM4) on the ICD weakened the inhibitory effect of RIC-3 on the functional surface expression of the channels. We infer that in Xenopus oocytes, RIC-3 reduction of functional surface expression of 5HT3A receptors is mediated by binding to the two identified binding sites.

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.

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.

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.

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 recombinant glycine receptor fragment forms homo-oligomers distinct from its GABAA counterpart

The ligand-gated ion channel receptor superfamily includes receptors for glycine, GABA, acetylcholine and serotonin. Whereas the acetylcholine and serotonin receptors mediate excitory neurotransmissions, both glycine and GABAA receptors are inhibitory. In this study, a fragment of the human glycine receptor α1 subunit, consisting of residues Ala165-Met291 (numbering based on the precursor protein), was hyperexpressed for the first time in Escherichia coli. This fragment is highly homologous in sequence to the corresponding fragment of the GABAA receptor. The recombinant fragment was found to have stable β-rich secondary structure, similar to that found for the homologous GABAA receptor fragment, and ordered tertiary packing, suggesting a stable structural domain. Results from laser scattering studies suggest that the fragment forms trimers in solution. In addition, SDS-induced changes in secondary structure were found to occur prior to changes in oligomerization status, suggesting that oligomerization was secondary structure dependent. A study of quaternary structure using single particle analysis electron microscopy (EM) also suggested that the fragment formed homo-trimers. One trimer measures approximately 7.5 nm in diameter with a central cavity approximately 1.5 nm across. This is the first EM study on a single domain of the glycine receptor and the result is in contrast to the pentameric assembly of the equivalent GABAA receptor fragment reported by us earlier. The fact that this fragment alone could form oligomers in vitro suggests that amino acid residues within this segment may be involved in the oligomerization of the glycine receptor in vivo. Furthermore, the finding that two cousin receptor fragments form distinct quaternary structures indicates that sequence similarity does not necessarily imply quaternary structure similarity and, hence, care must be taken when applying a structure model derived from studies of individual receptors to the whole ligand-gated ion channel superfamily.

(-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor.

Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the alpha 7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2. 2]octane-3,5'-oxazolidin-2'-one] (4a), a potent full agonist at the rat alpha 7 nicotinic receptor, which is highly selective for the rat alpha 7 nicotinic receptor over the alpha 4 beta 2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the alpha 7 nicotinic receptor affinity.

Identification of a New Ligand Binding Domain in the α1 Subunit of the Inhibitory Glycine Receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed "loop A" ligand binding domain. Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, 193A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, ImaX values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile93 and Asn102, as contributing to the four-loop model of ligand binding.

Heritable Mutations in the Glycine, GABAA/ and Nicotinic Acetylcholine Receptors Provide New Insights into The Ligand-Gated Ion Channel Receptor Superfamily

Publisher Summary This chapter highlights new set of information that has become available with the identification of heritable disease-causing mutations in various ligand-gated ion channel (LGIC) receptor subunits. The chapter summarizes the collective state of the structural and functional knowledge of the LGIC receptor superfamily. Comparison of the sites of heritable mutations with existing knowledge of the structure and function of the ligand gated ion channel receptors predicts the likely consequences of some heritable mutations. Whereas others, particularly the mutations that cause hyperekplexia in the glycine receptor, have defined new receptor domains involved in the process of signal transduction. The significance of these findings is that they provide a means of developing a unified model of the mechanism of action of the ligand-gated ion channel receptors. Excitatory signals are mediated by neurotransmitters such as acetylcholine, whereas inhibitory signals are mediated by glycine or γ-aminobutyric acid (GABA). The identification of naturally occurring heritable mutations in the various members of the LGIC receptor superfamily has provided a new set of targets for structure-function analysis. Mutations in subunits of the glycine receptor lead to a variety of startle syndromes in man and mouse. Mutations in subunits of the nicotinic acetylcholine receptor cause several forms of congenital myasthenia gravis and a rare form of epilepsy, whereas GABA A receptor subunit mutations result in insecticide resistance and alcohol sensitivity.

Therapeutic Potential of Serotonin 5-HT3 Antagonists in Neuropsychiatric Disorders

Serotonin 5-HT3-receptors are the only monoamine neurotransmitter receptors that are a member of the ligand-gated ion channel receptor superfamily, enabling these receptors to modulate fast synaptic transmission.

Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor.

The receptor for the inhibitory neurotransmitter glycine is a member of the ligand-gated ion channel receptor superfamily. Point mutations in the gene encoding the alpha 1 subunit of the glycine receptor-channel complex (GlyR) have recently been identified in pedigrees with the autosomal dominant neurological disorder, startle disease (hyperekplexia). These mutations result in the substitution of leucine or glutamine for arginine 271. This charged residue is located near the ion channel region and is predicted to affect chloride permeation through the GlyR. We found little evidence for this role from the anion/cation selectivity and lack of pronounced rectification of currents flowing through recombinant human alpha 1 subunit GlyRs containing the startle disease mutations. We reveal, however, that the startle disease mutations profoundly disrupt GlyR function by causing 230-410-fold decreases in the sensitivity of receptor currents activated by the agonist glycine. Additionally, we report corresponding 56- and 120-fold reductions in the apparent binding affinity (Ki) of glycine to the mutant GlyRs, but no change in the binding affinity of the competitive antagonist, strychnine. Thus, startle disease reduces the efficacy of glycinergic inhibitory neurotransmission by producing GlyRs with diminished agonist responsiveness. Our results show that startle disease mutations define a novel receptor activation site.

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily.

Rat pituitary cells selectively express mRNA encoding the short isoform of the γ2 GABA A receptor subunit

The GABA A receptor belongs to the ligand-gated ion channel receptor superfamily and appears to be composed of from 4 to 5 subunits which interact with each other. Molecular cloning of cDNAs encoding the different subunits of the GABA A receptor has revealed an unexpected heterogeneity which includes at least 4 homologous classes of subunits: these classes, designated as α, β, γ and δ contain multiple variants. We have measured the steady-state levels of mRNAs encoding different (α1, α4, β1, β2, β3, γ2 long, γ2 short and δ) GABA A receptor subunits in the rat anterior pituitary and cerebellum using a polymerase chain reaction (PCR)-derived method. We found that pituitary cells express mRNAs encoding the α1, β1 and β3 GABA A receptor subunits whereas the transcripts for α4, β2 and δ were undetectable. We also found that pituitary cells selectively express the short isoform of the γ2 subunit mRNA. These data indicate that the expression of the various GABA A receptor subunits is cell-specific and support the concept that the diversity in function and pharmacology of the GABA A receptor is based on the ability of cells to specify the expression of selective GABA A receptor subunits.