Concatenated nicotinic acetylcholine receptors: A gift or a curse?
ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are vital for normal mammalian brain function. Cys-loop receptors are pentameric ligand-gated ion channels formed from five identical or homologous subunits oriented around a central ion-conducting pore, which result in homomeric or heteromeric receptors, respectively. Within a given Cys-loop receptor family, many different heteromeric receptors can assemble from a common set of subunits, and understanding the properties of these heteromeric receptors is crucial for the continuing quest to generate novel treatments for human diseases. Yet this complexity also presents a hindrance for studying Cys-loop receptors in heterologous expression systems, where full control of the receptor stoichiometry and assembly is required. Therefore, subunit concatenation technology is commonly used to control receptor assembly. In theory, this methodology should facilitate full control of the stoichiometry. In reality, however, we find that commonly used constructs do not yield the expected receptor stoichiometries. With ternary or more complex receptors, concatenated subunits must assemble uniformly in only one orientation; otherwise, the resulting receptor pool will consist of receptors with mixed stoichiometries. We find that typically used constructs of ?4?2 nAChR dimers, tetramers, and pentamers assemble readily in both the clockwise and the counterclockwise orientations. Consequently, we investigate the possibility of successfully directing the receptor assembly process using concatenation. We begin by investigating the three-dimensional structures of the ?4?2 nAChR. Based on this, we hypothesize that the minimum linker length required to bridge the C terminus of one subunit to the N terminus of the next is shortest in the counterclockwise orientation. We then successfully express receptors with a uniform stoichiometry by systematically shortening linker lengths, proving the hypothesis correct. Our results will significantly aid future studies of heteromeric Cys-loop receptors and enable clarification of the current contradictions in the literature.
Project description:?-aminobutyric acid type A receptors (GABAARs), the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are arguably the most challenging member of the pentameric Cys-loop receptors to study due to their heteromeric structure. When two or more subunits are expressed together in heterologous systems, receptors of variable subunit type, ratio, and orientation can form, precluding accurate interpretation of data from functional studies. Subunit concatenation is a technique that involves the linking of individual subunits and in theory allows the precise control of the uniformity of expressed receptors. In reality, the resulting concatemers from widely used constructs are flexible in their orientation and may therefore assemble with themselves or free GABAAR subunits in unexpected ways. In this study, we examine functional responses of receptors from existing concatenated constructs and describe refinements necessary to allow expression of uniform receptor populations. We find that dimers from two commonly used concatenated constructs, ?-23-? and ?-10-?, assemble readily in both the clockwise and the counterclockwise orientations when coexpressed with free subunits. Furthermore, we show that concatemers formed from new tetrameric ?-10-?-?-? and ?-10-?-?-? constructs also assemble in both orientations with free subunits to give canonical ??? receptors. To restrict linker flexibility, we systematically shorten linker lengths of dimeric and pentameric constructs and find optimized constructs that direct the assembly of GABAARs only in one orientation, thus eliminating the ambiguity associated with previously described concatemers. Based on our data, we revisit some noncanonical GABAAR configurations proposed in recent years and explain how the use of some concatenated constructs may have led to wrong conclusions. Our results help clarify current contradictions in the literature regarding GABAAR subunit stoichiometry and arrangement. The lessons learned from this study may guide future efforts in understanding other related heteromeric receptors.
Project description:Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.
Project description:We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose ?4 and ?2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (?4)(3)(?2)(2)vs. (?4)(2)(?2)(3). Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (?4)(3)(?2)(2) exceeds that of (?4)(2)(?2)(3). The simulations account for published data on transfected Neuro2a cells in which ?4?2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (?4)(3)(?2)(2) stoichiometry to mostly (?4)(2)(?2)(3). The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (?4)(2)(?2)(3) and (?4)(3)(?2)(2) populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.
Project description:The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.
Project description:Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Native GABAA receptors are heteromeric assemblies sensitive to many important drugs, from sedatives to anesthetics and anticonvulsant agents, with mutant forms of GABAA receptors implicated in multiple neurological diseases. Despite the profound importance of heteromeric GABAA receptors in neuroscience and medicine, they have proven recalcitrant to structure determination. Here we present the structure of a tri-heteromeric ?1?1?2SEM GABAA receptor in complex with GABA, determined by single particle cryo-EM at 3.1-3.8 Å resolution, elucidating molecular principles of receptor assembly and agonist binding. Remarkable N-linked glycosylation on the ?1 subunit occludes the extracellular vestibule of the ion channel and is poised to modulate receptor assembly and perhaps ion channel gating. Our work provides a pathway to structural studies of heteromeric GABAA receptors and a framework for rational design of novel therapeutic agents.
Project description:Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus.We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling.The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(-)-4-amino-3-hydroxybutyric acid [R(-)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > ?-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(-)- and S(+)-GABOB.The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.
Project description:The ligand binding site of Cys-loop receptors is formed by residues on the principal (+) and complementary (-) faces of adjacent subunits, but the subunits that constitute the binding pocket in many heteromeric receptors are not yet clear. To probe the subunits involved in ligand binding in heteromeric human 5-HT(3)AB receptors, we made cysteine substitutions to the + and - faces of A and B subunits, and measured their functional consequences in receptors expressed in Xenopus oocytes. All A subunit mutations altered or eliminated function. The same pattern of changes was seen at homomeric and heteromeric receptors containing cysteine substitutions at A(R92) (- face), A(L126)(+), A(N128)(+), A(I139)(-), A(Q151)(-) and A(T181)(+), and these receptors displayed further changes when the sulphydryl modifying reagent methanethiosulfonate-ethylammonium (MTSEA) was applied. Modifications of A(R92C)(-)- and A(T181C)(+)-containing receptors were protected by the presence of agonist (5-HT) or antagonist (d-tubocurarine). In contrast modifications of the equivalent B subunit residues did not alter heteromeric receptor function. In addition a double mutant, A(S206C)(-)(/E229C)(+), only responded to 5-HT following DTT treatment in both homomeric and heteromeric receptors, indicating receptor function was inhibited by a disulphide bond between an A+ and an A- interface in both receptor types. Our results are consistent with binding to an A+A- interface at both homomeric and heteromeric human 5-HT(3) receptors, and explain why the competitive pharmacologies of these two receptors are identical.
Project description:To study characterization of zebrafish glycine receptors (zGlyRs), we assessed expression and function of five ?- and two ß-subunit encoding GlyR in zebrafish. Our qPCR analysis revealed variable expression during development, while in situ hybridizations uncovered expression in the hindbrain and spinal cord; a finding consistent with the reported expression of GlyR subunits in these tissues from other organisms. Electrophysiological recordings using Xenopus oocytes revealed that all five ? subunits form homomeric receptors activated by glycine, and inhibited by strychnine and picrotoxin. In contrast, ß subunits only formed functional heteromeric receptors when co-expressed with ? subunits. Curiously, the second transmembranes of both ß subunits were found to lack a phenylalanine at the sixth position that is commonly associated with conferring picrotoxin resistance to heteromeric receptors. Consistent with the absence of phenylalanines at the sixth position, heteromeric zGlyRs often lacked significant picrotoxin resistance. Subsequent efforts revealed that resistance to picrotoxin in both zebrafish and human heteromeric GlyRs involves known residues within transmembrane 2, as well as previously unknown residues within transmembrane 3. We also found that a dominant mutation in human GlyR?1 that gives rise to hyperekplexia, and recessive mutations in zebrafish GlyRßb that underlie the bandoneon family of motor mutants, result in reduced receptor function. Lastly, through the use of a concatenated construct we demonstrate that zebrafish heteromeric receptors assemble with a stoichiometry of 3?:2ß. Collectively, our findings have furthered our knowledge regarding the assembly of heteromeric receptors, and the molecular basis of ß subunit-conferred picrotoxin resistance. These results should aid in future investigations of glycinergic signaling in zebrafish and mammals.
Project description:The alpha4beta2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The alpha4 and beta2 nAChR subunits assemble into two alternate stoichiometries, (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2), which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn2+. We show that Zn2+ exerts an inhibitory modulatory effect on (alpha4)(2)(beta2)(3) receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (alpha4)(3)(beta2)(2) receptors. Furthermore, Zn2+ inhibition on (alpha4)(2)(beta2)(3) nAChRs is voltage-dependent, whereas it is not on (alpha4)(3)(beta2)(2) receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn(2+). Zn(2+) inhibition is mediated by a site located on the beta2(+)/alpha4(-) subunit interfaces on both receptor stoichiometries. alpha4(H195) and beta2(D218) are key determinants of this site. Zn2+ potentiation on (alpha4)(3)(beta2)(2) nAChRs is exerted by a site that resides on the alpha4(+)/alpha4(-) of this receptor stoichiometry. alpha4(H195) on the (-) side of the ACh-binding alpha4 subunit and alpha4(E224) on the (+) side of the non-ACh-binding alpha4 subunit critically contribute to this site. We also identified residues within the beta2 subunit that confer voltage dependency to Zn2+ inhibition on (alpha4)(2)(beta2)(3), but not on (alpha4)(3)(beta2)(2) nAChRs.
Project description:BACKGROUND AND PURPOSE:Anion-selective Cys-loop receptors (GABA and glycine receptors) provide the main inhibitory drive in the CNS. Both types of receptor operate via chloride-selective ion channels, though with different kinetics, pharmacological profiles, and localization. Disequilibrium in their function leads to a variety of disorders, which are often treated with allosteric modulators. The few available GABA and glycine receptor channel blockers effectively suppress inhibitory currents in neurons, but their systemic administration is highly toxic. With the aim of developing an efficient light-controllable modulator of GABA receptors, we constructed azobenzene-nitrazepam (Azo-NZ1), which is composed of a nitrazepam moiety merged to an azobenzene photoisomerizable group. EXPERIMENTAL APPROACH:The experiments were carried out on cultured cells expressing Cys-loop receptors of known subunit composition and in brain slices using patch-clamp. Site-directed mutagenesis and molecular modelling approaches were applied to evaluate the mechanism of action of Azo-NZ1. KEY RESULTS:At visible light, being in trans-configuration, Azo-NZ1 blocked heteromeric ?1/?2/?2 GABAA receptors, ?2 GABAA (GABAC ), and ?2 glycine receptors, whereas switching the compound into cis-state by UV illumination restored the activity. Azo-NZ1 successfully photomodulated GABAergic currents recorded from dentate gyrus neurons. We demonstrated that in trans-configuration, Azo-NZ1 blocks the Cl-selective ion pore of GABA receptors interacting mainly with the 2' level of the TM2 region. CONCLUSIONS AND IMPLICATIONS:Azo-NZ1 is a soluble light-driven Cl-channel blocker, which allows photo-modulation of the activity induced by anion-selective Cys-loop receptors. Azo-NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination.