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 this 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 probably 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. 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.
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).
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 reaction catalyzed by LIC family members is:
ions (in) ↔ ions (out)