1.A.9 The Neurotransmitter Receptor, Cys loop, Ligand-gated Ion Channel (LIC) Family
Members of the LIC family of ionotropic neurotransmitter receptors (also called the cys-loop ligand gated ion channel family because of the signature cysteine loop in the amino terminal domain) are found in vertebrate and invertebrate animals and prokaryotes (TC# 1.A.9.8.1; Bocquet et al., 2007; Sine and Engel, 2006; Thompson et al., 2010). Because of the extracellular N-terminal ligand-binding domain, they exhibit receptor specificity for (1) acetylcholine (AcCh), (2) serotonin, (3) glycine, (4) glutamate and (5) γ-aminobutyric acid (GABA)in vertebrates. All of these receptor channels are hetero- or homopentameric. The best characterized are the nicotinic acetylcholine receptors (nAChRs) which are pentameric channels of α2βγδ (immature muscle) nα2βγδ (mature muscle; see 1.A.9.1.1) (Witzemann et al., 1990; Wada et al., 1998; Khiroug et al., 2002) subunit composition. All subunits are homologous and have four transmembrane α-helices, M1-M4. In all members of this family, each of the five subunits has four transmembrane alpha-helices (M1-M4) with M2 lining the pore, M1 and M3 associate with M2, and M4 is outermost and adjacent to the membrane lipids. M4 has a variety of roles: its interaction with neighboring M1 and M3 helices is important for receptor assembly, it provides transmit information on the lipid content of the membrane to the gating mechanism, it forms a vital link to the extracellular domain via the Cys-loop, and it influences channel activity (da Costa Couto et al. 2020).
nAChRs are cation-selective ligand-gated ion channels exhibiting variable Ca2+ permeability depending on their subunit composition (Fucile 2017). Inhaled anesthetics alter the conformational states of LICs by binding within discrete cavities that are lined by portions of the four TMSs (Solt et al. 2006). Heusser et al. 2018 have argued in favor of a multisite model of transmembrane allosteric modulation by anesthetics, including a possible link between lipid- and receptor-based theories, that could inform the development of new anesthetics. Propofol Is an allosteric agonist with multiple binding sites on concatemeric ternary GABAA receptors (Shin et al. 2018). Alpha1beta3delta receptors may share stoichiometry and subunit arrangements with alpha1beta3gamma2 GABAA receptors (Feng and Forman 2018). 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH) is a potent inhibitor of neuronal nicotinic receptors (Papke et al. 2005). Determinants of charge selectivity of the Cys-loop family of ligand-gated ion channels have been reviewed (Jensen et al. 2005). The prokaryotic forms may function either as anion-selective or cation-selective channels, playing a role as chemotactic receptors for low molecular weight solutes (Tasneem et al. 2005).
Zn2+-activated cation channels of vertebrates and glutamate/serotonin-activated anion channels and GABA-gated cation channels of invertebrates are also in this family (Chen et al., 2006). Ligand binding has been reported to open the channel by a cis-trans prolylisomerization event (Cymeset al., 2005; Lummis et al., 2005). An intra-membrane proton binding site has been linked to activation of a bacterial LIC (Wang et al., 2011). Agonists activate alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site distinct from the AcCholine site (Gill et al., 2011). Molecular mechanisms of alcohol modulation (Rothberg 2012), desensitization (Keramidas and Lynch 2012) and assembly (Tsetlin et al., 2011) of nicotinic and other Cys-loop receptors have been reviewed. Acidic residues on the sides of the channel mouth and in the extracellular domain play a role in cationic selectivity (Colón-Sáez and Yakel 2013). Anesthetic binding occurs in common transmembrane domains of several of the LIC recpetors (Kinde et al. 2016).
Function based analysis of major biological pathways and mechanisms associated with schizophrenia (SCZ) genes identified glutaminergic receptors (e.g., GRIA1, GRIN2, GRIK4, GRM5), serotonergic receptors (e.g., HTR2A, HTR2C), GABAergic receptors (e.g., GABRA1, GABRB2), dopaminergic receptors (e.g., DRD1, DRD2), calcium-related channels (e.g., CACNA1H, CACNA1B), and solute transporters (e.g., SLC1A1, SLC6A2) (Sundararajan et al. 2018). Others are involved in neurodevelopment (e.g., ADCY1, MEF2C, NOTCH2, SHANK3). Biological mechanisms involving synaptic transmission, regulation of the membrane potential and transmembrane ion transport were identified as leading molecular functions associated with SCZ genes (Sundararajan et al. 2018).
The 5-HT(3) and acetylcholine receptors (cationic ion channels) and the GABA(A) and glycine receptors (anionic ion channels) generally depolarize or hyperpolarize, respectively, the neuronal membrane. Within the amino-terminal extracellular region, all members of this family exhibit a similar architecture of ligand binding domains, and a number of key residues are completely conserved (Connolly, 2008). Zhu and Hummer (2009) have concluded that the conformational transition from open and closed states involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition. N-alcohols potential H+-activated LIC currents in prokaryotes and eukaryotes (including glycine, GABA and acetylcholine receptors) (Howard et al., 2011).
The three-dimensional structures of the protein complex in both the open and closed configurations have been solved (Miyazawa and Unwin, 2003; Baenziger and Corringer, 2011). The five subunits (each of 400-500 amino acyl residues in length) are arranged in a ring with their 'M2' transmembrane helical spanners lining the central channel. The five M2 segments come together in the middle of the membrane to form the channel gate, and the gate opens upon binding of acetylcholine to distant sites in the N-terminal domains of the two α-subunits. The M2 segment determines the anion versus cation selectivity (Menard et al., 2005). These general structural features are probably valid for all members of the family. The acetylcholine receptor subunit consists of two domains, the channel domain and the ligand-binding extracellular domain. The latter is homologous to a soluble protein, the acetylcholine binding protein (AChBP), the structure of which has been solved at high resolution (Brejc et al., 2001). Nicotine interacts with nicotinic acetylcholine receptors and decreases food intake through activation of POMC neruons (Mineur et al. 2011). An intrasubunit nicotinic acetylcholine receptor binding site for the positive allosteric modulator Br-PBTC has been identified (Norleans et al. 2019).
Pentameric ligand-gated ion channels of the Cys-loop family mediate fast chemo-electrical transduction. Bocquet et al., 2009 presented the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation (TC #1.A.9.9.1), shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analyses of GLIC and ELIC revealed, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation (Bocquet et al., 2009).
According to Miyazawa et al. (2003), the pore is shaped by the inner ring of 5 α-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 α-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighboring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart (Miyazawa et al., 2003). (Nury et al., 2010) have presented a microsecond molecular dynamics simulation of channel gating in a nicotinic receptor homologue.
Bouzat et al. (2004) have identified structural requirements for functionally coupling the AcCh binding domain to the pore-forming domain. At least three loops in the AcCh binding domain interact with the pore domain to trigger channel opening. Modeling suggests a network of interacting loops between the two domains mediate allosteric coupling (Bouzat et al., 2004). Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ('Phi blocks') move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening, is comprised of residues near the transmitter binding site, and the second is comprised of residues near the base of the extracellular domain (Purohit and Auerbach, 2007).
Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. Single-molecule kinetic analysis has been used to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the α-subunit of the murine acetylcholine receptor (Purohitet al., 2007). αM2 move in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.
GABA interacts with three kinds of receptors, classes A, B and C. Classes A and C receptors are ligand-gated Cl- channels while class B receptors activate other channels via G proteins. GABA binding to both Class A and C receptors opens the Cl- channels, leading to increased membrane conductance. These two classes of receptors differ in their antagonist specificities and therefore are distinguished pharmacologically. These receptors consist of 6 types of subunits: α, β, γ, δ, ε, and π. There are 6 αs, 3 βs and 3 γs, and 1 each of the δ, ε and π subunits. Usually the pentamer consists of 2αs, 2 βs and 1 γ, each with 4 putative TMSs, a long N-terminus and a short C-terminus, both extracellular. These receptors possess binding sites for anti-epileptic drugs, sedatives and anesthetics. Evidence suggests that the TMS1-2 hairpin loops of the 5 subunits comprise the GABA receptor pore (Filippova et al., 2004). GABA type A receptors, the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. They bind at intersubunit sites (Chiara et al. 2013).
γ-Aminobutyric acid type A (GABAA) receptors consist of subunits whose assembly forms a neurotransmitter-gated anion channel. Subunits for this receptor constitute a large family whose members are classified according to primary structure as α, β, γ, and δ, ε, π, and ρ subunits. Recombinant expression studies of different α variants in combination with a β variant and the γ2 subunit demonstrated that GABAA receptors with distinct pharmacological properties are generated. A distinctive mark of these αxβxγ2 receptors is their ability to bind modulatory compounds such as benzodiazepines (BZs), which can modulate γ-aminobutyric acid (GABA)-gated channel activity at an allosteric site. The α variants determine the affinity of GABAA receptor subtypes toward these modulatory compounds, and members of the γ-subunit class, in particular, the γ2 variant, are essential for the architecture of the BZ site. Furthermore, the γ subunits impart a large unitary conductance on GABAA channels (Herb et al., 1992). N
Nineteen GABAAR subunits had been identified and categorized into eight classes by 2018,, alpha1-6, beta1-3, gamma1-3, delta, epsilon, theta, pi and rho1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., alpha6, beta2 and gamma2) (Has and Chebib 2018). The subunits within each class have an aa sequence identiy of 70% or more, whereas those with a sequence identity of 30% or less are grouped into different classes. There is a wide range of subunit combinations (Has and Chebib 2018).
The endocannabinoid system is a lipid signaling network that modulates different brain functions. Sigel et al. (2011) showed a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation (Sigel et al., 2011).
γ-aminobutyric type A (GABAA ) receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics, and anesthetics. Pyrazoloquinoline 2-p-methoxyphenylpyrazolo [4,3-c] quinolin-3(5H)-one (CGS 9895) is a positive allosteric modulator acting through the alpha+/beta- interface in the extracellular domain of GABAA receptors. The alpha1 Y209 residue present at the extracellular alpha+/beta- subunit interface is a key residue for the positive allosteric modulation of the GABAA receptor by CGS 9895 (Maldifassi et al. 2016). Neurosteroid inhibitors such as pregnenolone sulphate bind from the lipid bilayer to sites that are distinct from those of the GABA channel blocker picrotoxinin, and different GABAARs are differentially affected by these inhibitors (Seljeset et al. 2018).
γ2 and δ subunits share approximately 35% sequence identity with α and β subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The γ2 subunit is the rat homologue of the human γ2 subunit shown to be important for benzodiazepine pharmacology (Shivers et al., 1989). Functional GABA receptors mediating Cl- uptake in a picrotoxin-sensitive process (α5, β1, γ1) have been identified and shown to be functional in renal proximal tubular cells (Sarang et al., 2008).
A novel type of GABA receptor has been characterized (Beg and Jorgensen, 2003). It is an excitatory GABA gated cation channel (TC #1.A.9.7.1). This channel is the EXP-1 receptor of the nematode, C. elegans. It is as divergent in sequence from the A and C class anion selective GABA receptors as it is from the other ligand-gated ion channel proteins in TCDB. It therefore represents a new subfamily in the LIC family. Inhibitory glycine receptors (GlyR) mediate Cl- influx and bind strychnine with nanomolar affinity. They are present in various nervous tissues (spinal cord, brain stem, caudal brain and retina (Cascio, 2004). Reduced activities of mutants often results in channelopathies involving muscle tone regulation including human startle disease (hyperekplexia). There are multiple subunit subtypes in humans (α1, α2, α3, α4 and β subunits) (Cascio, 2004)). Alternative splicing also occurs. In adults, the most common pentameric form consists of α1and β subunits. The M2α-helix region peptide of α1 GlyR in lipid vesicles forms chloride-conducting pores, and sympathetic M2-based peptides form Cl- channels in cell membranes (Mitchell et al., 2000).
There are seven classes of serotonin (5 hydroxytryptamine (5HT) receptors, six of which are G-protein linked, plus one of which is a homo- or heteropentameric ligand gated non-specific cation channel (TC #1.A.9.2.1 and 2) of the LIC family (Reeves and Lummis, 2002). Anion selective serotonin receptors may be present in C. elegans (TC #1.A.9.6.1). As for other LIC family members, four transmembrane hydrophobic segments (TMSs) (M1-M4) are predicted by hydropathy analysis.
The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] mediates rapid excitatory responses in peripheral and central neurons by activating ligand-gated ion channels (5-HT3 receptors). These receptors are expressed in a variety of peripheral ganglia, where they are thought to modulate responses to pain, and to control reflexes of the enteric and cardiovascular systems. In the central nervous system, 5-HT3 receptors have been implicated in the control of emesis, and antagonists of 5-HT3 receptors have found clinical use for suppression of the nausea that accompanies postoperative recovery and many cancer therapies. Most families of ligand-gated ion channels are composed of multiple subunit types that assemble in alternative combinations to achieve functional diversity (Hanna et al., 2000).
Recombinant expression of 5-HT3A subunits alone yields functional 5-HT3receptors, but, heteromeric assemblies of the human 5-HT3A and 5-HT3B subunits more closely resemble native 5-HT3 receptors of rodent ganglia with respect to their single-channel conductance and permeability to Ca2+ ions. Consequently, it is likely that rodent ganglia normally express heteromeric 5-HT3 receptors. However, the 5-HT3 receptors of different species display several distinctive properties, particularly with respect to their pharmacological profiles (Hanna et al., 2000).
The channel protein complexes of the LIC family preferentially transport cations or anions depending on the channel (e.g., the acetylcholine and serotonin receptors are cation-selective while glycine receptors are anion-selective). α1β heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in adult mammals. (Burzomato et al., 2004). Glycine binding increases Cl-conductance producing superpolarization and inhibition of neuronal firing. Gephyrin (Q9NQX3) anchors glycine receptors to subsynaptic microtubules.
Several homologous bacterial LIC family members have been identified (eg, 1.A.9.8-9) Hilf and Dutzler (2008) have presented X-ray structures of a pentameric ligand-gated ion channel from the bacterium Erwinia chrysanthemi at 3.3 A resolution. This high resolution structure provides a model system for the investigation of the general mechanisms of ion permeation and gating within the LIC family.
Pentameric ligand-gated ion channels (LGICs) are fast-gating receptors, represented by cationic nicotinic acetylcholine (nAChR) and serotonin (5HT3R) receptors, and by anionic GABA and glycine (GlyR) receptors. Because of a highly conserved sequence of 13 amino acids flanked by two canonical cysteine residues shared by all members of the family, these receptors are also known as the Cys-loop family. These receptors are allosteric transmembrane proteins made of five identical or non-identical subunits arranged (pseudo) symmetrically around a central ion pore in the membrane. In nAChR, upon ACh binding, the receptor interconverts into discrete allosteric states, with each state corresponding to a different physiological state: resting (closed), active (open), and desensitized (closed) (Grutter et al. 2006).
The inhibitory glycine receptor is a ligand-gated ion channel with a pentameric assembly from ligand binding alpha and structural beta subunits. In addition to alpha subunit gene variants (alpha1-alpha4) and developmental alterations in subunit composition of the receptor protein complex, alternative splicing of both alpha and beta subunits has been found to contribute to glycine receptor heterogeneity (Oertel et al. 2007).
Ca2+ permeability is determined by charged amino acids at the extracellular end of the M2 transmembrane domain, which could form a ring of amino acids at the outer end of the cation channel. Alpha4, alpha5, and beta3 subunits all have a homologous glutamate in M2 that contributes to high Ca2+ permeability, whereas beta2 has a lysine at this position. Subunit combinations or single amino acids changes at this ring that have all negative charges or a mixture of positively and negatively charged amino acids are permeable to Ca2+. All positive charges in the ring prevent Ca2+ permeability. Increasing the proportion of negative charges is associated with increasing permeability to Ca2+ (Tapia et al. 2007).
Hibbs and Gouaux (2011) presented the three-dimensional structure of an inhibitory anion-selective Cys-loop receptor, the homopentameric Caenorhabditis elegans glutamate-gated chloride channel α (GluCl), at 3.3 Å resolution. The structures were determined with the allosteric agonist ivermectin (PDB#3RIF), the neurotransmitter L-glutamate and the open-channel blocker picrotoxin. Ivermectin, used to treat river blindness, binds in the transmembrane domain of the receptor and stabilizes an open-pore conformation. Glutamate binds in the classical agonist site at subunit interfaces, and picrotoxin directly occludes the pore near its cytosolic base. GluCl provides a framework for understanding mechanisms of fast inhibitory neurotransmission and allosteric modulation of Cys-loop receptors in general.
Cys loop, glutamate, and P2X receptors are ligand-gated ion channels (LGICs) with 5, 4, and 3 protomers, respectively. Agonists and competitive antagonists apparently induce opposite motions of the binding pocket (Du et al., 2012). Agonists, usually small, induce closure of the binding pocket, leading to opening of the channel pore, whereas antagonists, usually large, induce opening of the binding pocket, thereby stabilizing the closed pore.
Using crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment, structural motifs that are conserved within the nAChR family, contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, and also bacterial homologues have been analyzed. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TMS4 ring in conveying the influence of the lipid membrane environment to the middle TMS1-TMS3 ring and this, in turn, to the inner TMS2 channel-lining ring, which determines ion selectivity.
LICs or pLGICs have the same overall structure but with different combinations of agonist specificities and permeant ion charge selectivities. Three distantly related cation-selective members, the mouse muscle nicotinic acetylcholine receptor (nAChR), and the bacterial GLIC and ELIC channels, have differing sensitivities to TMA+ and TEA+ which block the nAChR and GLIC but not ELIC at low concentrations which transports both cations. Lidocaine binding speeds up the current-decay time courses of nAChR and GLIC in the presence of saturating concentrations of agonists, but its binding to ELIC slows this time course. Thus, one can not generalize results obtained with one channel to others (Gonzalez-Gutierrez and Grosman 2015).
Acetylcholine binds in the nAChR extracellular domain at the interface between two subunits and a large number of nAChR-selective ligands, including agonists and competitive antagonists, that bind at the same site are known. More recently, ligands that modulate nAChR function by binding to sites located in the transmembrane domain have been studied. These include positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent allosteric modulators (SAMs) and compounds that are able to activate nAChRs via an allosteric binding site (allosteric agonists) (Chatzidaki and Millar 2015).
The amine-gated Erwinia chrysanthemi LIC is activated by primary amines, while the transmembrane domain of the Gloeobacter violaceus LIC is activated by protons. Schmandt et al. 2015 found that a chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines revealed a closed-pore conformation and an C-terminal domain that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels.
Cys-loop receptors have structural elements that are well conserved with a large extracellular domain (ECD) harboring an alpha-helix and 10 beta-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures (Langlhofer and Villmann 2016). Except the TMS 3-4 loop, their lengths are only 7-14 residues. The TMS 3-4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all of these homologous receptors. The ICD is defined by the TMS 3-4 loop together with the TMS 1-2 loop preceding the ion channel pore (Langlhofer and Villmann 2016). Crystallization has revealed structures for some members of the CLR family, but to allow crystallization, the intracellular loop was usually replaced by a short linker present in prokaryotic CLRs, so their structures as not known. Nevertheless, this intracellular loop appears to function in desensitization, modulation of channel physiology by pharmacological substances, and posttranslational modifications. Motifs important for trafficking are therein, and the ICD interacts with scaffold proteins enabling inhibitory synapse formation (Langlhofer and Villmann 2016).
Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate-gated Cl- channels in parasites. It is known that IVM binds to the transmembrane domains of several ligand-gated channels, such as Cys-loop receptors and P2X receptors. Chen et al. 2017 found that the G-protein-gated inwardly rectifying K+ (GIRK) channel is also activated by IVM directly.
Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand gated ion channels and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids suggest the presence of multiple binding sites in these receptors. This has been demonstrated using GLIC (TC# 1.A.9.8.1) as the target channel protein (Cheng et al. 2018).
GABA(A) receptors are ligand-gated ion channels consisting of five subunits from eight subfamilies, each assembled in four hydrophobic transmembrane domains. This pentameric structure not only allows different receptor binding sites, but also various types of ligands, such as orthosteric agonists and antagonists, positive and negative allosteric modulators, and second-order modulators and non-competitive channel blockers. There are both synthetic and natural GABA(A)-receptor modulators. Çiçek 2018 reviewed natural GABA(A)-receptor modulators and discussed their structure-activity relationships.
Cell surface expression of type-A GABA receptors (GABAARs) is a critical determinant of the efficacyfor inhibitory neurotransmission. Pentameric GABAARs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as rho1 and beta3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as alpha and gamma, are retained within intracellular compartments. Normally, surface expression of alpha and gamma subunits requires co-assembly with beta subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Hannan and Smart 2018 identified two residues in the transmembrane domains of alpha and gamma subunits, which, when substituted for their rho1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the rho1 transmembrane residues for the alpha-subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABAAR trafficking and signaling. Although not ligand-gated, alpha and gamma homomeric ion channels were functional at the cell surface (Hannan and Smart 2018).
The protein has two cholesterol binding sites: an intersubunit site between TM3 and TM1 of adjacent subunits and an intrasubunit site between TM1 and TM4. In both sites, cholesterol is oriented such that the 3OH group points toward the center of the transmembrane domains rather than toward either the cytosolic or extracellular surfaces(Budelier et al. 2019). Allopregnanolone, a neurosteroid that allosterically modulates pLGICs, binds to the same binding pockets although the binding orientations of the two ligands were different, with the 3OH group of allopregnanolone pointing to the intra- and extracellular termini of the TMSs rather than to their centers. Cholesterol increases, whereas allopregnanolone decreases the thermal stability of GLIC. Thus, cholesterol and neurosteroids bind to common hydrophobic pockets in GLIC, but their effects depend on the orientation and specific molecular interactions unique to each sterol. I
Ionized side chains - whether pore-facing or buried - in the first α-helical turn of the second TMS determines charge discrimination in the substrate ion. However, electrostatics of backbone atoms are not critically involved. On the basis of electrophysiological observations, not only the sign of charged side chains but also their conformations seem to be crucial determinants of cation-anion selectivity. Thus, side-chain conformation is important for charge selectivity in Cys-loop receptors (Harpole and Grosman 2019).
At nM concentration, APPsα is an allosteric activator of α7-nAChR, mediated by C-terminal 16 amino acids (CTα16) (Korte 2019). At µM concentrations, Rice et al. 2019 identified the GABABR1a as a target of APPsα, binding the sushi 1 domain via a 17–amino acid sequence (17-mer). These receptors activate opposing downstream cascades.
CLIC from a Desulfofustis deltaproteobacterium (TC# 1.A.9.9.3), the x-ray structure of which has been determined, includes a periplasmic NTD fused to the conventional ligand-binding domain (LBD) (Hu et al. 2020). The NTD consists of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface is associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+. DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) should be reclassified as a 'locally closed' conformation. Structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs (Hu et al. 2020).
The reaction catalyzed by LIC family members is:
ions (in) ↔ ions (out)
Nicotinic acetylcholine-activated cation-selective channel, pentameric α2βγδ (immature muscle) nα2βγδ (mature muscle). A combination of symmetric and asymmetric motions opens the gate, and the asymmetric motion involves tilting of the TM2 helices (Szarecka et al. 2007). Acetylcholine receptor δ subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita (Brownlow et al., 2001; Webster et al., 2012). Residues in TMS2 and the cytoplasmic loop linking TMSs 3 and 4 influence conductance, selectivity, gating and desensitization (Peters et al., 2010). nAChR and TRPC channel proteins (1.A.4) mediate nicotine addiction in many animals from humans to worms (Feng et al., 2006). Cholesterol recognition motifs in transmembrane domains of the human nicotinic acetylcholine receptor have been identified (Baier et al., 2011). Allosteric modulators of the α4β2 subtype of neuronal nicotinic acetylcholine receptors, the dominant type in the brain, are numerous (Pandya and Yakel, 2011). α2β2 and α4β2 nicotinic acetylcholine receptors are inhibited by the β-amyloid(1-42) peptide (Pandya and Yakel, 2011b). The A272E mutation in the alpha7 subunit gives rise to spinosad insensitivity without affecting activation by acetylcholine (Puinean et al. 2012). Inhibited by general anaesthetics (Nury et al., 2011). The X-ray crystal structures of the extracellular domain of the monomeric state of human neuronal alpha9 nicotinic acetylcholine receptor (nAChR) and of its complexes with the antagonists methyllycaconitine and alpha-bungarotoxin have been determined at resolutions of 1.8 A, 1.7 A and 2.7 A, respectively (Zouridakis et al. 2014). Structurally similar allosteric modulators of α7 nAChR exhibit five different pharmacological effects (Gill-Thind et al. 2015). Mutations causing slow-channel myasthenia show that a valine ring in the channel is optimized for stabilizing gating (Shen et al. 2016). Quinoline derivatives act as agonists or antagonists depending on the type and subunit (Manetti et al. 2016). Conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a ""bubble"" that collapses to initiate ion conduction (Gupta et al. 2016). A high-affinity cholesterol-binding domain has been proposed for this and other ligand-gated ion channels (Di Scala et al. 2017). Positive allosteric modulators have been identified (Deba et al. 2018). Menthol stereoisomers exhibit fifferent effects on alpha4beta2 nAChR upregulation and dopamine neuron spontaneous firing (Henderson et al. 2019). Corticosteroids exert direct inhibitory action on the muscle-type AChR (Dworakowska et al. 2018). Both deltaL273F and epsilonL269F mutations impair channel gating by disrupting hydrophobic interactions with neighboring alpha-subunits. Differences in the extent of impairment of channel gating in delta and epsilon mutant receptors suggest unequal contributions of epsilon/alpha and delta/alpha subunit pairs to gating efficiency (Shen et al. 2019). Diffusion dynamics of the gangliosides, GM1s and AChRs is uniformly affected by the intracellular ATP level of a living muscle cell (He et al. 2020).
Acetylcholine receptors of Homo sapiens α2βγδ or ε
The nicotinic acetylcholine receptor alpha 6 isoform 1 of 505 aas and 6 or 7 putative TMSs, with one N-terminal TMS, one C-terminal TMS, and 4 or 5 centrally located TMSs. 66% identical to TC# 1.A.9.1.6. A 3 aa deletion in the transmembrane domain causes resistance to spinosad, a macrocyclic lactone insecticide (Wang et al. 2016). Mutations in the orthologous α6 subunit of Rhyzopertha dominica (lesser grain borer; 81% identical to the moth protein) also gave rise to spanosad resistance (Wang et al. 2018).
AcChR of Plutella xylostella (Diamondback moth) (Plutella maculipennis)
Acetylcholine-activated cation-selective channel, alpha-type, Acr-16 of 504 aas and 6 putative TMSs. Four negative allosteric modulators of this channel in the parasite have been identified (Zheng et al. 2016).
Acr-16 of Ascaris suum (Pig roundworm) (Ascaris lumbricoides)
Nicotinic acetylcholine receptor with three subunits, non-alpha subunit ShAR2beta of 545 aas, as well as two additional "non-alpha subunits of 714 and 736 aas, respectively, all with 6 TMSs, 1 N-terminal, 4 central, and 1 C-terminal (Bentley et al. 2007).
Trimeric nAcChR of Schistosoma haematobium (Blood fluke)
Neuronal acetylcholine receptor with two subunits, α- and β-subunits, Unc-63 (Lev7; 502 aas) and Acr-2 (575 aas), respectively. Probably acts in cholinergic motoneurons to
regulate presynaptic neurotransmitter release, thereby ensuring normal
level of excitation of cholinergic motoneurons during locomotion
(Jospin et al. 2009). Involved in nAChR sensitivity to nicotine and levamisole (Culetto et al. 2004; Gottschalk et al. 2005). The AcChR subunits in C. elegans have been compared with those of parasitic nematodes (Holden-Dye et al. 2013).
Neuronal AcChR of Caenorhabditis elegans
Acetylcholine receptor with two subunits, α and β, Deg-3 (564 aas)and Acr-4 (548 aas). Subunits of the non-synaptic neuronal AChR, which may play a role in chemotaxis towards choline. After binding choline or acetylcholine, the AChR responds by an extensive change in conformation that affects all subunits and leads to opening of an ion-conducting channel across the plasma membrane (Treinin et al. 1998; Yassin et al. 2001).
AcChR of Caenorhabditis elegans
Acr-16 subunit of a levamisole-insensitive nicotinic receptor of 498 aas (Touroutine et al. 2005). C. elegans has 32 AcChR subunits, 22 of them of the alpha-type, and these are divided into at least five classes, DEG-3-like (9), ACR-16- like (11), UNC-8-like (3), UNC-38-like (3) and Unc-29-like (4) (Holden-Dye et al. 2013).
ACR-16 of Caenorhabditis elegans
Beta-subunit (Unc-29; 493 aas) of a nicotinic AcChR. Non-alpha subunit of nAChR involved in nAChR sensitivity to nicotine and levasimole (Gottschalk et al. 2005).
UNC-29 of Caenorhabditis elegans
Nicotinic acetylcholine receptor, Eat-2/Eat-18 in the MC
pharyngeal motor neuron involved in pharyngeal pumping. It plays a role in
the determination of life span, possibly via calorific restriction (McKay et al. 2004; Huang et al. 2004). Eat-18 may be the CRE-EAT-18 protein with TC# 8.A.47.1.3.
Eat2/Eat18 of Caernorhabditis elegans
Neuronal acetylcholine receptor subunit alpha-5 of 429 aas and 4 apparent TMSs. It is part of an alpha-bungarotoxin binding acetylcholine receptor (Wu et al. 2005).
ACHA5 of Bactrocera dorsalis (oriental fruit fly)
Acetyl choline binding protein, AchBP, of 229 aas, corresponding to the N-terminal extracellular domain of AcChRs. The crystal structure is known (Lin et al. 2016). It modulates synaptic transmission (Smit et al. 2001). This soluble protein has enhanced our understanding of the requirements for agonistic and antagonistic interactions at the ligand recognition site of the nAChRs. Camacho-Hernandez and Taylor 2020 have reviewed the potential and limitations of soluble surrogates, termed the AChBP family, in drug development.
AchBP of Lymnaea stagnalis (great pond snail) (Helix stagnalis)
The nicotinic acetylcholine activated cation selective channel precursor, Acr-2 or Acr-3/Unc-38 (both β and α-type chains are required for activity; levamisole-gated; activity reduced by antagonists mecamylamine and d-tubocurarine) (Squire et al., 1995; Baylis et al., 1997). nAChR and TRPC channel proteins (1.A.4) mediate nicotine addiction in many animals from humans to worms (Feng et al., 2006). Functions at synapses in the nervous system and at neuromuscular junctions (Towers et al. 2006). Neonicottinoides affect worm behavior and development (Kudelska et al. 2017). C. elegans has a large number of nAcChR genes, only some of which are retained in parasitic nematodes (Holden-Dye et al. 2013). RIC-3 is an nAcChR chaparone (Treinin 2008).
Acr-2 or Acr-3/Unc-38 of Caenorhabditis elegans
Acr-2 (β) (P48182)
Acr-3 (β) (Q93149)
Unc-38 (α) (Q23022)
Nicotinic acetylcholine receptor, nAChR subunit type B of 527 aas and 4 TMSs (Jiao et al. 2019).
nAChR of Lymnaea stagnalis (Great pond snail) (Helix stagnalis)
Nicotinic acetylcholine receptor, nAChR with 4 subunits, Alpha1, 2, and 3 as well as beta1. Bradysia odoriphaga is a destructive insect pest, damaging more than 30 crop species. Nicotinic acetylcholine receptors (nAChRs) mediating fast excitatory transmission in the central nervous systems in insects are the molecular targets of some economically important insecticides including imidacloprid, which has been widely used to control B. odoriphaga in China since 2013. Shan et al. 2020 cloned seven nAChR subunit genes from B. odoriphaga, including Boα1, Boα2, Boα3, Boα7, Boα8, Boβ1 and Boβ3. They resemble the Drosophila melanogaster nAChR alpha1 subunit, including an extracellular N-terminal domain containing six functional loops (loop A-F), a signature Cys-loop with two disulfide bond-forming cysteines separated by 13 amino acid residues, and four typical TMSs 1 - 4) in the C-terminal region. Four of these subunits are included in TCDB.
nAChR of Bradysia odoriphaga
Alpha9/alpha10 (α9α10) neuronal acetylcholine receptor with the two subunits of 450 aas (α9; Chrna9 or NACHRA9) and 479 aas (α10; Chrna10 or NACHRA10). It is an ionotropic receptor with a probable role in the modulation of auditory stimuli. Agonist binding induces a conformation change that leads to the opening of an ion-conducting channel across the plasma membrane (Sgard et al. 2002, Zouridakis et al. 2014). The channel is permeable to a range of divalent cations including calcium, the influx of which may activate a potassium current which hyperpolarizes the cell membrane (Zouridakis et al. 2014). In the ear, this may lead to a reduction in basilar membrane motion, altering the activity of auditory nerve fibers and reducing the range of dynamic hearing. This may protect against acoustic trauma, and may also regulate keratinocyte adhesion (Nguyen et al. 2000). Hair cell alpha9alpha10 nicotinic acetylcholine receptor functional expression is regulated by ligand binding and deafness gene products (Gu et al. 2020). Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10nAChRs which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Thus, the mechanism links cholinergic input with α9α10 assembly, identifies functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders (Gu et al. 2020).
Alpha9/alpha10 (α9α10) neuronal acetylcholine receptor of Homo sapiens
Nicotinic acetylcholine receptor β-1 subunit , Accβ1 (a target of insecticides (Yu et al., 2011; Tricoire-Leignel and Thany 2010)).
Accβ1 of Apis cerana (F6JX92)
Nicotinic acetylcholine receptor β-2 subunit, Accβ2 (a target of insecticides)
Accβ2 of Apis cerana (F6JVF4)
Acr-5 of Caenorhabditis elegans
The α4β2 nicotinic acetylcholine receptor. The NMR structure of the transmembrane domain and the multiple anaesthetic binding sites are known (Bondarenko et al., 2012). Mutations cause autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE; Díaz-Otero et al. 2000).
Nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy and for neuromuscular blocking agents used during surgery. This system contributes to cognitive functioning through interactions with multiple neurotransmitter systems and is implicated in various CNS disorders, i.e., schizophrenia and Alzheimer's disease. It provides an extra layer of molecular complexity by existing in two different stoichiometries determined by the subunit composition. By potentiating the action of an agonist through binding to an allosteric site, positive allosteric modulators can enhance cholinergic neurotransmission (Grupe et al. 2015). Most pentameric receptors are heteromeric. Morales-Perez et al. 2016 presented the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. The side chains of alpha4 L257 (9') and alpha4L264 (16') may beresponsible for the main constrictions in the transmembrane pore (Yu et al. 2019). Mechanistic steps for communication proceed (1) through a signal generated via loop C in the principal subunit, (2) transmitted gradually and cumulatively to loop F of the complementary subunit, and (3) to the TMSs through the M2-M3 linker (Oliveira et al. 2019).
α4β2 NAChR of Homo sapiens
The alpha7 (α-7) nicotinic acetylcholine receptor (alpha-7 nAcChR) of 502 aas is encoded by the CHRNA7 gene. Acetylcholine binding induces conformational changes that result in open channel formation; opening is blocked by α-bungarotoxin. The protein is a homopentamer. It interacts with RIC3 for proper folding and assembly. The nAChR, but not the glycine receptor, GlyR, exhibits hydrophobic gating (Ivanov et al. 2007). Low resolution NMR structures with associated anesthetics have been reported (Bondarenko et al. 2013). Allosteric modulators exhibit up to 5 distinct pharmacological effects (Gill-Thind et al. 2015). Based on pore hydration and size, a high resolution structure for the channel in the open conformation has been proposed (Chiodo et al. 2015). Agonists reduce dyskinesias in both early- and later-stage Parkinson's disease (Zhang et al. 2015). Monoterpenes inhibit the alpha7 receptor in the order: carveol > thymoquinone > carvacrol > menthone > thymol > limonene > eugenole > pulegone = carvone = vanilin. Among the monoterpenes, carveol showed the highest potency (Lozon et al. 2016). A revised structural model has been proposed (Newcombe et al. 2017). In humans, exons 5-10 in CHRNA7 are duplicated and fused to the FAM7A genetic element, giving rise to the hybrid gene CHRFAM7A. Its product, dupalpha7, is a truncated subunit lacking part of the N-terminal extracellular ligand-binding domain and is associated with neurological disorders, including schizophrenia, and immunomodulation (Lasala et al. 2018). alpha7 and dupalpha7 subunits co-assemble into functional heteromeric receptors, in which at least two alpha7 subunits are required for channel opening. Dupalpha7's presence in the pentameric arrangement does not affect the duration of the potentiated events. Using an alpha7 subunit mutant, activation of (alpha7)2(dupalpha7)3 receptors occurs through ACh binding at the alpha7/alpha7 interfacial binding site (Lasala et al. 2018). B-973 is an efficacious type II positive allosteric modulator (PAM) of alpha7 nicotinic acetylcholine receptors that, like 4BP-TQS and its active isomer GAT107, is able to produce direct allosteric activation in addition to potentiation of orthosteric agonist activity, which identifies it as an ago-PAM (Quadri et al. 2018). DB04763, DB08122 and pefloxacin are antagonists (they are NAMs) while furosemide potentiated ACh responses (it is a Pam) (Smelt et al. 2018). At nM concentration, APPsα (amyloid precursor protein) is an allosteric activator of α7-nAChR, mediated by the C-terminal 16 amino acids (CTα16) (Korte 2019). At µM concentrations, Rice et al. 2019 identified the GABABR1a as a target of APPsα, binding the sushi 1 domain via a 17–amino acid sequence (17-mer). These receptors activate opposing downstream cascades. The intrasubunit cavity of the α7 AcChR is important for the activity of type II positive allosteric modulators while the ECD-TMD junction and intersubunit sites are probably important for the activity of type I positive allosteric modulators (Targowska-Duda et al. 2019). Flavonoids are positive allosteric modulators of alpha7 nicotinic receptors (Nielsen et al. 2019). Active and desensitized state conformations have been examined (Chiodo et al. 2018). Modulators are able to activate or deactivate a7 receptors via allosteric binding; they are called positive allosteric modulators (PAMs) or negative allosteric modulators (NAMs) (Al Rawashdah et al. 2019). Functional divergence related sites cluster in the ligand binding domain, the beta2-beta3 linker close to the N-terminal alpha-helix, the intracellular linkers between transmembrane domains, and the "transition zone" (Pan et al. 2019). A series of phosphonate-functionalized 1,2,3-triazoles are positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (Nielsen et al. 2020). The E-1' --> A-1' substitution at the cytoplasmatic selectivity filter strongly affects sodium and chloride permeation in opposite directions, leading to a complete inversion of selectivity. Thus, structural determinants for the observed cationic-to-anionic inversion reveal a key role of the protonation state of residue rings far from the mutation, in the proximity of the hydrophobic channel gate (Cottone et al. 2020).
The homomeric α7 acetylcholine receptor of Homo sapiens
Nicotinic receptor, nAChRalpha7, of 560 aas and 5 TMSs. The beta-amyloid protein (TC# 1.C.50.1.1) can activate the nAChRalpha7 receptor (Hassan et al. 2019).
Nicotinic receptor, nAChRalpha7, of Drosophila melanogaster
The cation-selective pentameric nicotinic acetylcholine receptor, nAChR, with α (461 aas; P02710), β (493 aas; P02712), γ (506 aas; P02714) and δ (522 aas; P02718) subunits. The transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting or desensitized state (Sun et al. 2016). Studies with this receptor have been reviewed (Unwin 2013). Many small molecules interact with nAChRs including d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various more recently discovered compounds of different structural classes also interact with nAChRs including the low-molecular weight alkaloids, pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR (Kudryavtsev et al. 2015). Dimethylaniline mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine (Alberola-Die et al. 2016). Cholesterol is a potent modulator of the Torpedo nAChR (Baenziger et al. 2017). Cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel (Unwin 2017). 11beta-(p-azidotetrafluorobenzoyloxy)allopregnanolone (F4N3Bzoxy-AP), a general anesthetic, a photoreactive allopregnanolone analog and a potent GABAAR PAM,was used to characterize steroid binding sites in the Torpedo nAChR in its native membrane environment (Yu et al. 2019). The steroid-binding site in the nAChR ion channel was identified, and additional steroid-binding sites could also be occupied by other lipophilic nAChR antagonists. Structural features of the αM4 TMS determine how lipid dependent changes in alphaM4 structure may ultimately modify nAChR function (Thompson et al. 2020).
nAChR of Tetronarce californica (Pacific electric ray) (Torpedo californica)
Cyc-loop anion ligand-gated receptor of 453 aas and 6 TMSs, LIC1 (Mukherjee 2015).
LIC1 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
Uncharacterized ligand-gated ion channel of 539 aas and 4 TMSs.
Uncharacterized LIC of Chlorella variabilis (Green alga)
Serotonin (5-hydroxytryptamine)-activated cation-selective receptor/channel, 5-HT3R. Residues in TMS2 and the cytoplasmic loop linking TMSs 3 and 4 influence conductance, selectivity, gating and desensitization (Peters et al., 2010; McKinnon et al., 2011). Resveratrol enhances ion currents (Lee et al., 2011). Rings of charge within the extracellular vestibule influence ion permeation (Livesey et al., 2011). Based on the 3-d structure, serotonin binding first induces distinct conformational fluctuations at the side chain of W156 in the highly conserved ligand-binding cage, followed by tilting-twisting movements of the extracellular domain which couple to the transmembrane TM2 helices, opening the hydrophobic gate at L260 and forming a continuous transmembrane water pathway (Yuan et al. 2016). There are 5 isoforms of 5-HT3A which include 5-HT3AB, 5-HT3AC, 5-HT3AD, and 5-HT3AE, all of which have similar but distinct pharmacological profiles compared to those of 5-HT3A receptors (Price et al. 2017). Trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA) is a potent agonist with behavior different from that of 5-HT (Gasiorek et al. 2016). Two serotonin-bound structures of the full-length 5-HT3A receptor in distinct conformations reveal the mechanism underlying channel activation (Basak et al. 2018). The trans-cis isomerization of a proline at the interface between the extracellular and transmembrane domain may be the switch between closed and open states of the channel (Crnjar et al. 2019). SR 57227A is the most commonly used 5-HT3 receptor agonist with the ability to cross the blood brain barrier (Nakamura et al. 2019). Picrotoxin antagonizes serotonin (5-HT)3 receptors in a subunit-dependent fashon (Das and Dillon 2005). It interacts directly with the chaparone protein, Ric-3, (TC# 8.A.71.1.1) (Pirayesh et al. 2019). A nanopore based on the 5-HT3 receptor channel (see TC# 1.A.9.2.1) responds to an electric field than induces wetting of the hydrophobic gate (Klesse et al. 2020).
Serotonin (5HT3) receptor (5HT3R) of Homo sapiens (P46098)
The 5-hydroxytryptamine (serotonin) receptor-3A receptor/cation-selective ion channel, 5-HT3AR, of 454 aas. The channel is activated by the binding of serotonin to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. A variety of compounds modulate agonist-evoked responses of 5-HT3ARs and other Cys-loop receptors by binding to distinct allosteric sites (Lansdell et al. 2014). Alternative intersubunit pathways may exist for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. An arginine triplet located in the intracellular domain may determine the characteristic low conductance properties of the channel (Di Maio et al. 2015). The 12 Å resolution structure of the protein in a lipid bilayer (cryo EM) reveals topological features (Kudryashev et al. 2016). A chimeric receptor consisting of the extracellular domain of the 5-HT3A receptor and the transmembrane domain of a prokaryotic homologue, ELIC has been constructed (Price and Lummis 2018). The resulting receptor responds to 5-HT. Partial agonists and competitive antagonists activate and inhibit the chimera. Examination of a range of receptor modulators including ethanol, thymol, 5-hydroxyindole, and 5-chloroindole suggest that these compounds act via the transmembrane domain, except for 5-hydroxyindole, which can compete with 5-HT at the orthosteric binding site (Price and Lummis 2018). The receptor has 4 TMSs, M1 - M4, and Y441 in M4 interacts with D238 in M1, W459 in M4 interacts with F144 in the Cys loop, and D434 in M4 interacts with R251 in M2 according to the residue numbering system of Mesoy et al. 2019. This suggests that M4 helicies in LIC receptors interact with other parts of these receptors differently. Amino acid residues involved in agonist binding, linked to channel gating, that are proximal to the transmembrane domain for halothane modulation have been identified (Kim et al. 2009). Microsecond-timescale simulations suggest 5-HT mediates preactivation of the 5-HT3A serotonin receptor (Guros et al. 2019). Minimal structural rearrangement of the cytoplasmic pore occur during activation (Panicker et al. 2004).
5HT3AR of Homo sapiens
Zinc-activated ligand-gated cation channel of 412 aas and 5 TMSs, ZACN; ZAC. Zac displays potencies and efficacies in the rank orders of H+>Cu2+>Zn2+ and H+>Zn2+>Cu2+, respectively. ZAC appears to be non-selectively permeable to monovalent cations, whereas Ca2+ and Mg2+ inhibit the channel (Trattnig et al. 2016).
Zac of Homo sapiens
Adult strychnine-sensitive glycine-inhibited chloride (anion selective) heteropentameric channel (GlyR; GLRA1) consisting of α1- and β-subunits (Cascio, 2004; Sivilotti, 2010). Ivermectin potentiates glycine-induced channel activation (Wang and Lynch, 2012). Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids have been identified (Yévenes and Zeilhofer, 2011). Different subunits contribute asymmetrically to channel conductances via residues in the extracellular domain (Moroni et al., 2011; Xiong et al., 2012). Dominant and recessive mutations in GLRA1 are the major causes of hyperekplexia or startle disease (Gimenez et al., 2012). Open channel 3-d structures are known (Mowrey et al. 2013). Desensitization is regulated by interactions between the second and third transmembrane segments which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker, picrotoxin (TC# 8.C.1), prevents desensitization (Gielen et al. 2015). The x-ray structure of the α1 GlyR transmembrane domain has been reported (Moraga-Cid et al. 2015), and residue S296 in hGlyR-alpha1 is involved in potentiation by Delta(9)-tetrahydrocannabinol (THC) (Wells et al. 2015). The structure has also been elucidated by cryo EM (Du et al. 2015) and by x-ray crystalography (Huang et al. 2015). The latter presented a 3.0 A X-ray structure of the human glycine receptor-alpha3 homopentamer in complex with the high affinity, high-specificity antagonist, strychnine. The structure allowed exploration of the molecular recognition of antagonists. Comparisons with previous structures revealed a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain. The GlyR beta8-beta9 loop is an essential regulator of conformational rearrangements during ion channel opening and closing (Schaefer et al. 2017). Association of GlyR with the anchoring protein, gephyrin (Q9NQX3), is due to a hydrophobic interaction formed by Phe 330 of gephyrin and Phe 398 and Ile 400 of the GlyR beta-loop (Kim et al. 2006). Alcohols and volatile anesthetics enhance the function of inhibitory glycine receptors (GlyRs) by binding to a single anaesthetic binding site (Roberts et al. 2006). Aromatic residues in the GlyR M1, M3 and M4 α-helices are essential for receptor function (Tang and Lummis 2018). The neurological disorder, startle disease, is caused by glycinergic dysfunction, mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies (Schaefer et al. 2018). GlyRs can be modulated by positive allosteric modulators (PAMs) that target the extracellular, transmembrane and intracellular domains (Lara et al. 2019). Mutations in GLRA1 give rise to hyperekplexia (Milenkovic et al. 2018). Neurosteroid binding sites of GABAARs are conserved in the GlyRs (Alvarez and Pecci 2019). The intracellular domain of homomeric glycine receptors modulates agonist efficacy (Ivica et al. 2020). Inhibitory glycinergic transmission in the adult spinal cord is primarily mediated by glycine receptors (GlyRs) containing the alpha1 subunit. Alpha1ins, a longer alpha1 variant with 8 amino acids inserted into the intracellular large loop between TMSs 3 and 4, is expressed in the dorsal horn of the spinal cord, distributed at inhibitory synapses, and it is engaged in negative control over nociceptive signal transduction. Activation of metabotropic glutamate receptor 5 (mGluR5; TC# 9.A.14.7.1) specifically suppressed alpha1ins-mediated glycinergic transmission and evoked pain sensitization. Extracellular signal-regulated kinase (ERK) was critical for mGluR5 to inhibit alpha1ins. By binding to a D-docking site created by the 8-amino-acid insert ERK catalyzed alpha1ins phosphorylation at Ser380, which favored alpha1ins ubiquitination at Lys379 and led to alpha1ins endocytosis. Disruption of the ERK interaction with alpha1ins blocked Ser380 phosphorylation, potentiated glycinergic synaptic currents, and alleviated inflammatory and neuropathic pain (Zhang et al. 2019). The startle disease mutation (αS270T) affects the opening state for activation of presynaptic homomeric GlyRs, as well as postsynaptic heteromeric GlyRs, but the former are affected more. Both respond to glycine less efficiently (Wu et al. 2020). Cannabinoids exert therapeutic effects on several diseases such as chronic pain and startle disease by targeting glycine receptors (GlyRs). They target a serine residue at position 296 in the third TMS of the alpha1/alpha3 GlyR on the outside of the channel at the lipid interface where cholesterol concentrates. GlyRs are associated with cholesterol/caveolin-rich domains. and cholesterol reduction significantly inhibits cannabinoid potentiation of glycine-activated currents (Yao et al. 2020).
Glycine receptor of heteromeric α1/ β-subunit channels (GlyR) of Homo sapiens
α1 chain (GlrA1) (P23415)
α2 chain (GlrA2) (P23416)
α3 chain (GlrA3) (O75311)
β chain (GlrB) (P48167)
Photoreceptor in large monopolar cells (LMCs) histamine-gated chloride channel, HclA (Ort) (forms homomers, and heteromers with HclB; homomers resemble native LMC receptors (Pantazis et al., 2008)). hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins (Iovchev et al. 2006).
HclA of Drosophila melanogaster (A1KYB4)
Glutamate receptor of 552 aas, GluCl-2 (Lynagh et al. 2014).
GluCl-2 of Schistosoma mansoni (Blood fluke)
Glutamate-inhibited chloride (anion-selective) channel, CIα chain. This protein is 98% identical to the ortholog in Musca domestica (the house fly). Fluralaner (Bravecto) is an isoxazoline ectoparasiticide which potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The amino acid, Leu315, in Musca GluCls is important in determining the selectivity of fluralaner and ivermectin which react in opposite ways (Nakata et al. 2017).
Glutamate receptor CIα chain of Drosophila melanogaster
Glutamate-gated chloride channel (GluClα or Glc-1) (α-subunits when mutated confer resistance to the antiparisitic drug, avermectin (ivermectin) (Dent et al., 2000)). A naturally occurring 4-aa deletion in the ligand binding domain of Glc-1 confers resistance to Avermectin (Ghosh et al., 2012). Several 3-d structures are known (3RIF; Hibbs and Gouaux, 2011). Ivermectin (avermectin; IVM), an anthelmintic drug, inhibits neuronal activity and muscular contractility in arthropods and nematodes, activating glutamate-gated chloride channels at nanomolar concentrations (Lynagh and Lynch, 2012; Calimet et al. 2013; Degani-Katzav et al. 2017). Ivermectin resistance has been studied in Haemonchus contortus (the Barber pole worm) leading to the conclusion that mutations to ivermectin resistance affected the intrinsic properties of the receptor with no specific effect on IVM binding (Atif et al. 2017). Glutamate binding triggers a rapidly reversible current in heteromeric channels formed by Glc-1 and Glc-2, while the anti-helmintic drug ivermectin and other avermectins trigger a permanently open channel configuration. Channels containing only Glc-1 are activated by ivermectin, but not by glutamate alon, and Glutamate binding triggers a rapidly reversible current in heteromeric channels formed by Glc-1 and Glc-2, while the anti-helmintic drug ivermectin and other avermectins including ibotenate trigger a permanently open channel configuration. Channels containing only Glc-1 are activated by ivermectin, but not by glutamate alone. The channel is blocked by picrotoxin and flufenamic acid (Cully et al. 1994; Das and Dillon 2005).
GluCl of Caenorhabditis elegans
Glutamate-gated chloride channel, GluC1 or Glc-4 (Yamaguchi et al., 2012). Ivermectin, an anthelminthic drug, inhibits neuronal activity and muscular contractility in arthropods and nematodes, activating glutamate-gated chloride channels at nanomolar concentrations (Lynagh and Lynch, 2012; Zemkova et al. 2014). Mutations in GluCl associated with field ivermectin-resistant head lice have been identified (Amanzougaghene et al. 2018).
GluC1 of Haemonchus contortus (P91730)
Glc-4 (GluC1) glutamate receptor of 500 aas. The x-ray structure of several states including two apo states have been solved, revealing the gating mechanism of cys-loop receptors (Althoff et al. 2014). Ligand-induced conformational gating has been proposed (Yoluk et al. 2015). Effects of L-glutamate, ivermectin, ethanol and anesthetics have been examined (Heusser et al. 2016).
Glc-4/GluC1 of Caenorhabditis elegans
Glutamate-gated chloride channel of 448 aas, GluCl. A point mutation, A309V in TMS 3, renders the protein and the organism > 11,000-fold resistant to abamectin, an insecticide of this moth, which is a global pest of cruciferous vegetables (Wang et al. 2015). Both A309V and G315E mutations contribute to target-site resistance to abamectin (Wang et al. 2017). Fluralaner (Bravecto) is an isoxazoline ectoparasiticide which potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The amino acid, Leu315, in Musca (fly) GluCls is important in determining the selectivity of fluralaner and ivermectin which react in opposite ways (Nakata et al. 2017).
GluCl of Plutella xylostella (Diamondback moth) (Plutella maculipennis)
Glutamate-gated chloride channel exon 3c variant of 447 aas and 5 TMSs. Okaramines produced by Penicillium simplicissimum AK-40 activate l-glutamate-gated chloride channels (GluCls) and thus paralyze insects. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely eliminated (Furutani et al. 2017).
GluCl of Bombyx mori (Silk moth)
Ligand-gated ion channel, Lgc-34 of 390 aas and 4 TMSs. IGDB-2, an Ig/FNIII protein, binds LGC-34 to control sensory compartment morphogenesis (Wang et al. 2017).
LGC-34 of Caenorhabditis elegans
γ-Aminobutyric acid (GABA)-inhibited chloride channel. The major central endocannabinoid, 2-arachidonoyl glycerol (2-AG), directly acts at GABA(A) receptors. It potentiates the receptor at low GABA concentrations (Sigel et al., 2011). Hydrophobic anions potently and uncompetitively antagonize GABA (A) receptor function (Chisari et al., 2011). Regulated by neurosteroids; activated by pregnenolone and allopregnenalone (Costa et al., 2012). Different subunits contribute asymmetrically to channel conductances via residues in the extracellular domain (Moroni et al., 2011). Potentiated by general anaesthetics (Nury et al., 2011). Direct physical coupling between the GABA-A receptor and the KCC2 chloride transporter underlies ionic plasticity in cerebellar purkinje neurons in response to brain-derived neurotrophic factor (BDNF) (Huang et al. 2013). GABA type A receptors, the brain's major inhibitory neurotransmitter receptors, are the targets for many general anesthetics, including volatile anesthetics, etomidate, propofol, and barbiturates. Anesthetics usually bind at intersubunit sites (Chiara et al. 2013). Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane beta+/alpha- inter-subunit interfaces. In heteromeric receptors, betaN265 (M2-15') on beta2 and beta3 subunits are important determinants of sensitivity to these drugs (Stewart et al. 2014). A P302L mutation in the gamma2 (γ2) subunit (Dravet syndrome in humans) of the mouse when expressed with the α1 and β3 subunits, produced a 90% decrease in conductance due to slow activation and enhance desensitization. It shifted the channel to a low-conductance state by reshaping the hour-glass-like pore cavity during transitions between closed, open, and desensitized states (Hernandez et al. 2017). Numerous postive and negative allosteric modulators have been identified (Maldifassi et al. 2016). Crystal structures of neurosteroids bound to alpha homopentameric GABAARs have revealed binding to five equivalent sites (Alvarez and Pecci 2018). Masiulis et al. 2019 reported high-resolution cryo-EM structures in which the full-length human alpha1beta3gamma2L GABAA receptor in lipid nanodiscs is bound to (1) the channel-blocker picrotoxin, (2) the competitive antagonist bicuculline, (3) the agonist GABA, and (4 AND 5) the classical benzodiazepines alprazolam and diazepam. They described the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region (Masiulis et al. 2019). Rare variants in the ε-subunit have been identified in patients with a wide spectrum of epileptic phenotypes (Markus et al. 2020).
GABA receptor of Rattus norvegicus
α-1 subunit precursor (P62813)
β-1 subunit precursor (P15431)
γ-1 subunit precursor (P23574)
δ subunit precursor (P18506)
ε subunit precursor (Q9ES14)
π subunit precursor (O09028)
ρ-1 subunit precurosr (O09028)
GABA associated (P60517) protein
GABA gated chloride channel of 537 aas and 4 TMSs, GABA or Rdl. The tropical cattle tick, Rhipicephalus microplus, is one of the most damaging parasites that affects cattle in tropical and subtropical regions in the world. Tick resistance to acaricides is dispersed worldwide, and a number of associated mutations in target site genes have been described. Phenylpyrazole (e.g., fipronil) and cyclodiene (e.g., lindane, dieldrin) insecticides both have the same mode of action, blocking the GABA-gated chloride channel encoded by the GABA-Cl gene. A conserved mutation, rdl (resistance to dieldrin) is found across a number of arthropods resistant to cyclodienes and phenylpyrazoles. In ticks, the mutation T290L, was identified in the second transmembrane (TMS2) domain of the GABA-gated chloride channel of Australian cattle tick populations that are resistant to dieldrin, but other mutations giving rise to resistance have been described. Cross-resistance between fipronil and lindane was reported in R. microplus populations (Castro Janer et al. 2019).
Rdl of Rhipicephalus microplus (Cattle tick) (Boophilus microplus)
γ-aminobutyric acid (GABA)-inhibited Cl- channel, type A (α-, β- γ-subunit precursors), regulated by GABA receptor accessory protein, GABARAP (Luu et al., 2006). The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator (Fisher 2009). The major central endocannabinoid, 2-arachidonoyl glycerol (2-AG), also directly acts at GABA(A) receptors to potentiate the receptor at low GABA concentrations (Sigel et al., 2011). The recpetor is also allosterically regulated by neurosteroids via TMS1 of the beta subunit (Baker et al. 2010). General anesthetic binding site(s) have been identified (Chiara et al., 2012; Woll et al. 2018). Hydrophobic anions potently and uncompetitively antagonize GABA (A) receptor function (Chisari et al., 2011). Regulated by neurosteroids; activated by pregnenolone and allopregnenalone (Costa et al., 2012). Allopregnanolone and its synthetic analog alphaxalone are GABAAR positive allosteric modulators (Yu et al. 2019). Different subunits contribute asymmetrically to channel conductances via residues in the extracellular domain (Moroni et al., 2011). Potentiated by general anaesthetics (Nury et al., 2011). Both the alpha and beta subunits are important for activation by alcohols and anaesthetics (McCracken et al. 2010). Direct physical coupling between the GABA-A receptor (of 4 TMSs) and the KCC2 chloride transporter underlies ionic plasticity in cerebellar purkinje neurons in response to brain-derived neurotrophic factor (BDNF) (Huang et al. 2013). An anesthetic binding site has been identified (Franks 2015). Desensitization is regulated by interactions between the second and third transmembrane segments which affect the ion channel lumen near its intracellular end. The GABAAR and GlyR pore blocker, picrotoxin (TC# 8.C.1), prevents desensitization (Gielen et al. 2015). The mechanism of action of methaqualone (2-methyl-3-O-tolyl-4(3H)-quinazolinone, Quaalude(R)), a sedative-hypnotic and recreational drug. Methaqualone is a positive allosteric modulator (PAM) at human alpha1,2,3,5beta2,3gamma2S GABAA receptors (GABAARs) expressed, whereas it displays diverse functionalities at the alpha4,6beta1,2,3delta GABAAR subtypes, ranging from inactivity (alpha4beta1delta), through negative (alpha6beta1delta) or positive allosteric modulation (alpha4beta2delta, alpha6beta2,3delta), to superagonism (alpha4beta3delta) (Hammer et al. 2015). The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Westergard et al. 2015 used functional experiments on alpha2beta1gamma2 GABAA receptors to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP showed competitive effects, supporting the presence of a T3 and ALLOP binding site at one or more subunit interfaces. Residues in the beta3 subunit, at or near the etomidate/propofol binding site(s), form part of the valerenic acid modulator binding pocket (Luger et al. 2015). IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, enhance GABAA receptor activation. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors (Forman and Miller 2016). Carisoprodol can directly gate and allosterically modulate type A GABA (GABAA) receptors (Kumar et al. 2017). The former sedative-hypnotic and recreational drug methaqualone (Quaalude) is a moderately potent, non-selective positive allosteric modulator of GABAA receptors (GABAARs) (Hammer et al. 2015). A methaqualone analog, 2-phenyl-3-(p-tolyl)quinazolin-4(3H)-one (PPTQ) exhibits intrinsic activity at micromolar concentrations and potentiates the GABA-evoked signaling at concentrations down to the low-nanomolar range (Madjroh et al. 2018). The PPTQ binding site is allosterically linked with sites targeted by neurosteroids and barbiturates. Anesthetic pharmacophore binding has been studied (Fahrenbach and Bertaccini 2018). GABAA receptors are modulated via several sites by GABA, benzodiazepines, ethanol, neurosteroids and anaesthetics among others. Amundarain et al. 2018 presented a model of the alpha1beta2gamma2 subtype GABAA receptor in the APO state and in complex with selected ligands, including agonists, antagonists and allosteric modulators. Sites in TMSs 2 and 3 are important for alcohol-induced conformational changes (Jung and Harris 2006). Many anesthetics and neurosteroids act through binding to the GABAAR transmembrane domainnad x-ray structures have revealed how α-xalone, a neurosteroid anaesthetic, binds and influences potentiation, activation and desensitization (Chen et al. 2018). AA29504 is an allosteric agonist and positive allosteric modulator of GABAA receptors (Olander et al. 2018). Allosteric shift analysis in mutant α1β3γ2L GABAA receptors indicates selectivity and cross-talk among intersubunit transmembrane anesthetic sites (Szabo et al. 2019). Several epilepsy-causing mutations have been identified in the genes of the α1, β3, and γ2 subunits comprising the GABAA receptor (Absalom et al. 2019). Constituents of the GABAA receptor include a transmembrane GARLH/LHFPL protein (TC# 1.A.82.1.7) and the inhibitory synaptic protein, neuroligin 2 (TC# 8.A.117.1.1) (Tomita 2019). GABAA receptors containing mutant alpha5 and alpha1 subunits all had reduced cell surface and total cell expression with altered endoplasmic reticulum processing, impaired synaptic clustering, reduced GABAA receptor function and decreased GABA binding potency. Thus, GABRA5 is a causative gene for early onset epileptic encephalopathy (Hernandez et al. 2019). Mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid (Sugasawa et al. 2019). Binding sites and interactions of propanidid and AZD3043 within GABAAR have been identified (Wang et al. 2018). Clptm1 limits GABAAR forward trafficking from the ER to the plasma membrane, and it regulates inhibitory homeostatic plasticity (Ge et al. 2018). The mechanisms of potentiation and inhibition of GABAA receptors by non-steroidal anti-inflammatory drugs, niflumic and mefenamic acids, have been described (Rossokhin et al. 2019). GABAARs are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). PAMs bind selectively to a single intersubunit site in the GABAAR transmembrane domain (Jayakar et al. 2019). The gamma2 subunit is required for clustering of these receptors, for recruitment of the submembrane scaffold protein gephyrin to postsynaptic sites, and for postsynaptic function of GABAergic inhibitory synapses (Alldred et al. 2005). The fourth TMS of the gamma2 subunit is required for postsynaptic clustering, but both the major cytoplasmic loop and the fourth transmembrane domain contribute to efficient recruitment of gephyrin to postsynaptic receptor clusters and are essential for restoration of miniature IPSCs (Alldred et al. 2005). Oligomerization and cell surface expression of recombinant GABAA receptors tagged in the delta subunit have been examined (Oflaz et al. 2019). The isoxazoline ectoparasiticide, fluralaner, exerts antiparasitic effects by inhibiting the function of GABARs, but substitutions of Gly333 in TMS3 led to substantial reductions in the sensitivity to fluralaner (Yamato et al. 2020). A potent photoreactive general anesthetic with novel binding site selectivity for GABAA receptors has been identified (Shalabi et al. 2020). GABAA receptor neurosteroid binding sites have been reviewed (Alvarez et al. 2019). Missense variants in GABRA2 are associated with early infantile epileptic encephalopathy (EIEE) as well as other disorders (Sanchis-Juan et al. 2020).
GABA type A receptor of Homo sapiens (α-/β-/γ-subunits + GABARAP)
α-subunit precursor (NP_000798)
β-subunit precursor (O18276)
γ-subunit precursor (NP_944494)
Gamma-aminobutyric acid (GABA) receptor alpha 2 subunit of 499 aas. It''s structure and sites of glycosylation and phsophorylation have been identified (Zuo et al. 2013).
Gamma-aminobutyric receptor alpha 2 subunit of Spodoptera litura (Asian cotton leafworm)
The GABA receptor consisting of α1, β3, and γ2 subunits. Heteropentameric receptor for GABA, the major inhibitory neurotransmitter in the vertebrate brain. Functions also as the histamine receptor and mediates cellular responses to histamine. Functions as a receptor for diazepines and various anesthetics, such as pentobarbital which bind to separate allosteric effector binding sites. Functions as ligand-gated chloride channel (Jayakar et al. 2015). GABRA1 mutations are associated with familial juvenile myoclonic epilepsy, sporadic childhood absence epilepsy, idiopathic familial generalized epilepsy, infantile spasms and Dravet syndrome. Thus, GABRA1 mutations are associated with infantile epilepsy including early onset epileptic encephalopathies including Ohtahara syndrome and West syndrome (Kodera et al. 2016).
GABA Receptor subunits α1/β3/γ2 of Homo sapiens
Human GABA-A (hGABA-A) rho1 receptor of 479 aas and 4 TMSs. The guanidine compound, amiloride, antagonized the heteromeric GABA-A, glycine, and nicotinic acetylcholine receptors, but it exhibits characteristics consistent with a positive allosteric modulator for the hGABA-A rho1 receptor (Snell and Gonzales 2016). Picrotoxinin binds to both GABAA-rho1 and -rho2 in the homomeric channels, but to GABAA-rho2 with 10x higher affinity (Naffaa and Samad 2016). The inhibitory site for ethanol in GABAA rho1 receptors regulates acute functional tolerance to moderate ethanol intoxication. Low sensitivity to alcohol intoxication is linked to risk for the development of alcohol dependence in humans (Blednov et al. 2017). Positive and negative allosteric modulators of GABAA receptors have been reviewed (Olsen 2018). A single amino acid change within the ion-channel domain accelerates desensitization and increases taurine agonism (Martínez-Torres and Miledi 2013).
GABA-A rho1 receptor of Homo sapiens
Gamma-aminobutyric acid receptor, LCCH3 of 496 aas and 4 TMSs/ GRD of 686 aas and 4 TMSs. LCCH3 combines with the ligand-gated ion channel subunit, GRD, to form cation-selective GABA-gated ion channels. The heteromultimeric receptor is activated by GABA (EC50=4.5 microm),
muscimol (EC50=4.8 microm) and trans-4-aminocrotonic acid (EC50=104.5
microm), and partially by cis-4-aminocrotonic acid (EC50=106.3 microm).
Picrotoxin effectively blocked the GABA-gated channel (IC50=0.25 microm), but bicuculline, TPMTA, dieldrin and lindane did
not. The benzodiazepines flunitrazepam and diazepam did not potentiate
the GABA-evoked current (Gisselmann et al. 2004). The system has been partially characterized from the small brown planthopper, Laodelphax striatellus (Fallen), a major
insect pest of crop systems in East Asia (Wei et al. 2017).
LCCH3/GRD of Drosophila melanogaster (Fruit fly)
GABA(A) receptor subunit alpha-3 of 492 aas and 4 TMSs, GABRA3. GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders. A loss of function presents a major pathomechanism. Loss increases the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern (Niturad et al. 2017). GABA, the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel.
GABRA3 of Homo sapiens
Rice stem borer GABA recpetor of 496 aas and 4 TMSs. Insect GABAR is a major targets of insecticides. cDNAs (CsRDL1A and CsRDL2S) encoding the two isoforms of RDL subunits were cloned from the rice stem borer Chilo suppressalis. Transcripts of both genes demonstrated similar expression patterns in different tissues and developmental stages, although CsRDL2S was approximately 2-fold more abundant than CsRDL1A throughout all development stages. Electrophysiological results using a two-electrode voltage clamp demonstrated that GABA activated currents in oocytes injected with both cRNAs. The EC50 value of GABA in activating currents was smaller in oocytes co-injected with CsRDL1A and CsRDL2S than in oocytes injected singly. The IC50 value of the insecticide fluralaner in inhibiting GABA responses was smaller in oocytes co-injected with different cRNAs than in oocytes injected singly. Co-injection also changed the potency of the insecticide dieldrin in oocytes injected singly. Thus, heteromeric GABARs were formed by the co-injections of CsRDL1A and CsRDL2S in oocytes. Although the presence of Ser at the 2'-position in the second TMS was responsible for the insensitivity of GABARs to dieldrin, this amino acid did not affect the potencies of the insecticides fipronil and fluralaner. Thus, C. suppressalis may adapt to insecticide pressure by regulating the expression levels of CsRDL1A and CsRDL2S and the composition of both subunits in GABARs.
GABAR of Chilo suppressalis (Asiatic rice borer moth)
GabaA1 receptor of 459 aas and 4 TMSs. 87% identical to the human homologue (TC# 1.A.9.5.4). Insecticides, abamectin, dieldrin, fluralaner and fipronil strongly inhibited GABA-induced inward current >50% at 10-6 M, while alpha-endosulfan, flufiprole and ethiprole inhibited <50% (Huang et al. 2018). Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae (Cao et al. 2019).
Lgc-53 of Caenorhabditis elegans (Q2PJ95)
The high affinity tyramine (amine-gated) chloride channel receptor, Lgc-55 (Ringstad et al., 2009). Activated by amphetamines (Safratowich et al. 2013).
Lgc-55 of Caenorhabditis elegans (Q9TVI7)
Lgc-40 of Caenorhabditis elegans (Q22741)
The prokaryotic H+-gated ion channel, GlvI or GLIC (Bocquet et al., 2007), solved at 2.9 Å resolution in the open pentameric state (3EHZ_E) (Bocquet et al., 2009; Corringer et al. 2010). The basis for ion selectivity has been reported (Fritsch et al., 2011). Two stage tilting of the pore lining helices results in channel opening and closing (Zhu and Hummer, 2010). The mechanical work of opening the pore is performed primarily on the M2-M3 loop. Strong interactions of this short and conserved loop with the extracellular domain are therefore crucial to couple ligand binding to channel opening. The H+-activated GLIC has an extracellular domain between TMSs M3 and M4 but lacks the intracellular domain (ICD) which is a distinct folding domain (Goyal et al., 2011). The structural basis for alcohol modulation of GLIC has been reported (Howard et al., 2011). The structure of the M2 TMS indicates that the charge selectivity filter is in the cytoplasmic half of the channel (Parikh et al. 2011). Below pH 5.0, GLIC desensitizes on a time scale of minutes. During activation, the extracellular hydrophobic region undergoes changes involving outward translational movement, away from the pore axis, leading to an increase in pore diameter. The lower end of M2 remains relatively immobile (Velisetty et al., 2012). During desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison to the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed-state (Velisetty et al. 2012). Pore opening and closing is well understood (Zhu and Hummer 2010). X-ray structures of general anaesthetics bound to GLIC reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer (Nury et al., 2011). Large blockers bind in the center of the membrane, but divalent transition metal ions bind to the narrow intracellular pore entry (Hilf et al., 2010). Alcohols and anaesthetics induce structural changes and activate ligand-gated ion channels of the LIC family by binding in intersubunit cavities (Sauguet et al. 2013; Ghosh et al. 2013). Gating at pH 4 has been visualized by x-ray crystallography (Gonzalez-Gutierrez et al. 2013) Site-directed spin labeling and x-ray analyses have revealed gating transition motions and mechanisms that distinguish active from desensitized states (Dellisanti et al. 2013; Sauguet et al. 2013). Gating involves major rearrangements of the interfacial loops (Velisetty et al. 2014). A single point mutation can change the effect of an anesthetic (desfurane; chloroform) from an inhibitor to a potentiator (Brömstrup et al. 2013). An interhelix hydrogen bond involving His234 is important for stabilization of the open state (Rienzo et al. 2014). The outermost M4 TMS makes distinct contributions to the maturation and gating of the related GLIC and ELIC homologs, suggesting that they exhibit divergent mechanisms of channel function (Hénault et al. 2015). The same allosteric network may underlie the actions of various anesthetics, regardless of binding site (Joseph and Mincer 2016). GLIC and ELIC (TC# 1.A.9.9.1) may represent distinct transmembrane domain archetypes (Therien and Baenziger 2017). Arcario et al. 2017 have demonstrate an anesthetic binding site in GLIC which is accessed through a membrane-embedded tunnel. The anesthetic interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel (Arcario et al. 2017). The gating mechanism has been studied (Lev et al. 2017). R-Ketamine inhibits members of the LIC family, and the structural and dynamics basis for the assymetric inhibitory modulation of ketamine has been revealed (Ion et al. 2017). Residue E35 has been identified as a key proton-sensing residue, as neutralization of its side chain carboxylate stabilizes the active state. Thus, proton activation occurs allosterically at the level of multiple loci with a key contribution of the coupling interface between the extracellular and transmembrane domains (Nemecz et al. 2017). General anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain (Fourati et al. 2018). GLIC's gating by protonation proceeds by making use of loop F, already known as an allosteric site in other pLGICs, instead of the classic orthosteric site (Hu et al. 2018). Binding of fentanyl to its binding site within GLIC results in conformational changes that inhibit conduction through the channel (Faulkner et al. 2019). This channel and others have been studied by high-speed atomic force microscopy (HS-AFM) which has made it possible to characterized the conformational dynamics of single unlabeled transmembrane channels and transporters (Heath and Scheuring 2019).
GlvI or GLIC of Gloeobacter violaceus (Q7NDN8)
Uncharacterized ligand-gated ion channel of 343 aas and 4 C-terminal TMSs.
LIC family protein of Lyngbya aestuarii
Ligand-gated ion channel of 312 aas and 5 TMSs, one N-terminal and 4 C-terminal (Jaiteh et al. 2016).
LIC of Thaumarchaeota archaeon N4
Uncharacterized ligand-gated ion channel of 351 aas and 4 TMSs.
UP of Francisella cf. novicida
The bacterial pentameric Cys-loop ligand-gated ion channel (Erwinia chrysanthemi ligand-gated ion channel), ELIC. A 3.3 Å resolution structure is available (Hilf and Dutzler, 2008; Corringer et al., 2010). X-ray analyses have identified three distinct binding sites for anaesthetics, one in the channel, one at the end of a TMS, and one in a hydrophobic pocket of the extracellular domain (Spurny et al. 2013). Motions involving desensitization have been defined (Dellisanti et al. 2013). Simulations indicate the similarities with and differences between the Acetylcholine receptor (Cheng et al. 2009). This family includes members with very divergent properties (Gonzalez-Gutierrez and Grosman 2015). Cysteamine is an agonist for ELIC (Hénault and Baenziger 2016). X-ray structures and functional measurements support a pore-blocking mechanism for the inhibitory action of short-chain alcohols which bind to the TMSs (Chen et al. 2016). GLIC (TC# 1.A.9.8.1) and ELIC may represent distinct transmembrane domain archetypes (Therien and Baenziger 2017), and both bind hopenoids at the mamalian cholesterol binding site (Barrantes and Fantini 2016). A high-resolution structure of ELIC in a lipid-bound state has revealed a phospholipid binding site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics (Hénault et al. 2019). This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. M4 is intrinsically flexible, and M4 deletions or disruptions of the lipid-binding site accelerate desensitization, suggesting that lipid interactions shape the agonist response (Hénault et al. 2019). 1-Palmitoyl-2-oleoyl phosphatidylglycerol (POPG) stabilizes the open state of ELIC relative to the desensitized state by direct binding to specific sites (Tong et al. 2019). The nicotinic acetylcholine receptor from the Torpedo electric organ, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity, and ELIC has a similar lipid sensitivity. Structures of ELIC in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoEM have been solved (Kumar et al. 2020). The largest differences occur at the level of loop C - at the agonist-binding sites - and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). The transmembrane pore is occluded similarly in both structures. POPC-only membranes prevent ECD-TMD coupling so that the "conformational wave" of liganded-receptor gating takes place in the ECD and the interfacial M2-M3 linker, but fails to penetrate the membrane and propagate into the TMD. The higher affinity for agonists, characteristic of the open- and desensitized-channel conformations, results from the tighter confinement of the ligand to its binding site; this limits the ligand's fluctuations, and thus delays its escape into bulk solvent (Kumar et al. 2020).
ELIC of Dickeya chrysanthemi (Pectobacterium chrysanthemi) (Erwinia chrysanthemi)
Cys-loop ligand-gated pentameric cation channel of 320 aas and 4 C-terminal TMSs sTeLIC (Hu et al. 2018); from a bacterial endosymbiont of Tevnia jerichonana (vent Tica). 28% identical to ELIC (TC# 1.A.9.9.1). The crystal structure has been determined in a wide open state, revealing a cavity for modulation. It is gated by alkaline pH. Two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions such as tetramethylammonium. Hu et al. 2018 also found that sTeLIC is allosterically potentiated by the aromatic amino acids, Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.
sTeLIC of a γ-proteobacterial endosymbiont of Tevnia jerichonana (vent Tica)
X-ray structures of CLIC of 680 aas and 1N-terminal and 4 C-terminal TMSs with a large N-terminal hydrophilic domain (NTD) from a Desulfofustis deltaproteobacterium have been solved. The protein includes a periplasmic NTD fused to the conventional ligand-binding domain (LBD) (Hu et al. 2020). The NTD consists of two jelly-roll domains interacting across each subunit interface. Binding of Ca2+ at the LBD subunit interface is associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca2+. DeCLIC crystallized in the absence of Ca2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) should be reclassified as a "locally closed" conformation. Structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca2+ modulation and periplasmic NTDs (Hu et al. 2020).
CLIC or pLGIC of Desulfofustis deltaproteobacterium