Nicotinic acetylcholine-activated cation-selective channel, pentameric α₂βγδ (immature muscle) nα₂βγδ (mature muscle), is activated by nicotine (Shen et al. 2022). 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). M4, the outermost helix, is involved in opening of the alpha4beta2 nACh receptor (Mesoy and Lummis 2020). Cholesterol modulates the organization of the gammaM4 transmembrane domain of the muscle nicotinic acetylcholine receptor (de Almeida et al. 2004). Cryo-EM images showed that cholesterol segregates preferentially around the constituent ion channel of the  receptor, interacting with specific sites in both leaflets of the bilayer. Cholesterol forms microdomains - bridges of rigid sterol groups that link one channel to the next (Unwin 2021).  Desnitro-imidacloprid (DN-IMI) functionally affects human neurons similarly to the well-established neurotoxicant nicotine by triggering activation of alpha7 and several non-alpha7 nAChRs (Loser et al. 2021).

Animals

Acetylcholine receptors of Homo sapiens α₂βγδ or ε
α (P02708)
β (P11230)
γ (P07510)
δ (Q07001)
ε (Q04844)

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). The Ascaris suum nicotinic acetylcholine receptor (nAChR) is modulated by compounds GSK575594A, diazepam and flumazenil (Stevanovic et al. 2021).

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 (474 aas and 4 TMSs in a 3 + 1 arrangement)/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).

Animals

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. The nicotinic acetylcholine receptor nAChR, is involved in immune regulation in pearl oysters (Pinctada fucata martensii). Neonicotinoids are selective modulators of insect nAChRs. These insecticides interact with the orthosteric sites of nAChRs, not only to activate nAChRs, but also to block the desensitizing component of nAChR responses. Recombinant vertebrate and insect/vertebrate hybrid nAChRs have been deployed to understand the mechanism of selectivity and diversity of neonicotinoid actions as well as to show that both alpha/alpha and alpha/non-alpha interfaces are involved in the interactions with neonicotinoids (Matsuda 2021).

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).  Point mutations in the nicotinic receptor alpha1 subunit can be responsible for slow-channel myasthenia (Kudryavtsev et al. 2021).

Alpha9/alpha10 (α9α10) neuronal acetylcholine receptor of Homo sapiens

Fusion protein with an N-terminal kinase domain (residues 1 - 268; homologous and 37% identical  to TC# 8.A.104.1.5) and a C-terminal acetylcholine receptor-α domain (residues 278 - 744, 45% identical to TC# 1.A.9.1.15) of C. elegans. These observations could reflect the presence of true fusion proteins, or they could be a result of sequencing errors.

Fusion protein of Halicephalobus sp. NKZ332

Nicotinic acetylcholine receptor β-1 subunit , Accβ1 (a target of insecticides (Yu et al., 2011; Tricoire-Leignel and Thany 2010)). 

Insects

Accβ1 of Apis cerana (F6JX92)

Nicotinic acetylcholine receptor β-2 subunit, Accβ2 (a target of insecticides)

Insects

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). A genetic variant of the nicotinic receptor α4-subunit causes sleep-related hyperkinetic epilepsy via increased channel opening (Mazzaferro et al. 2022).

Animals

α4β2 NAChR of Homo sapiens 
α4 (P43681)
β2 (P17787)

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 GABA_BR1a 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). Outer membrane mitochondrial nAChRs (e.g., α7 NAChR) regulate apoptosis-induced mitochondrial channel formation by modulating the interplay of apoptosis-related proteins (VDAC1 and Bax) in the mitochondrial outer membrane (Kalashnyk et al. 2020). PNU-120596, a positive allosteric modulator of mammalian alpha7 nicotinic acetylcholine receptor, increases the neuron response to alpha7 agonists while retarding desensitization (Vulfius et al. 2020). Differential interactions of resting, activated, and desensitized states of the alpha7 nicotinic acetylcholine receptor with lipidic modulators have been decumented (Zhuang et al. 2022).

Animals

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). Cholesterol content in the membrane promotes key lipid-protein interactions (Crnjar and Molteni 2021). Triple arginines are molecular determinants for pentameric assembly of the intracellular domain of 5-HT3A receptors (Pandhare et al. 2019). Five different subunits of the human serotonin (5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. The A and C subunits associate forming AC heteromer complexes at or near the cell surface, and a proportion can also form A or C homomers. Both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers (Abad et al. 2020). It is a biomarker for  endometriosis (EM), a common gynecological disorder that often leads to irregular menstruation and infertility (Jiang et al. 2022).Perić et al. 2022 have  summarized information on the location of the components of the serotonin system in the human placenta, their regulation, function, and alterations in pathological pregnancies.

Animals

Serotonin (5HT3) receptor (5HT3R) of Homo sapiens (P46098)

The heteromeric serotonin 5HT_3A receptor (Hanna et al., 2000). The influences of serotonin on single neurons, neural networks, and cortical circuits in the prefrontal cortex (PFC) of the rat is where the effects of serotonin have been most thoroughly studied (Puig and Gulledge 2011).

Animals

The 5HT_3A/5HT_3B receptor of Rattus norvegicus
5HT_3A (Q35563)
5HT_3B (Q9JJ16)

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). The intracellular domain starts with a short loop after the third TMS, followed by a short alpha-helical segment, a large unstructured loop, and finally, the membrane-associated MA-helix that continues into the last TMS (Stuebler and Jansen 2020). The MA-helices from all five subunits form the extension of the transmembrane ion channel and shape what has been described as a "closed vestibule," with the lateral portals obstructed by loops and their cytosolic ends forming a tight hydrophobic constriction. Although conformational changes associated with gating promote cross-linking for I409C/R410C, which in turn decreases channel currents, cross-linking of L402C/L403C is functionally silent in macroscopic currents. These results support the hypothesis that concerted conformational changes open the lateral portals for ion conduction, rendering ion conduction through the vertical portal unlikely (Stuebler and Jansen 2020).

Animals

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 is an atypical cys-loop receptor in terms of its identified agonists and channel characteristics, but its signal transduction seems to undergo similar conformational transitions as those in other members of the family (Madjroh et al. 2021). N-(thiazol-2-yl)-benzamide analogs comprise a class of selective antagonists of the ZAC (Madjroh et al. 2021).

Zac of Homo sapiens

The 5-hydroxytryptamine (serotonin) receptor 3B, HTR3B, of 441 aas and 4 or 5 TMSs in a 1 (N-terminal) + 2 or 3 (residues 240 - 320) + 1 TMS (C-terminal). This is one of the several different receptors for 5-hydroxytryptamine (serotonin), a biogenic hormone that functions as a neurotransmitter, a hormone, and a mitogen. This receptor is a ligand-gated ion channel, which when activated, causes fast, depolarizing responses. It is a cation-specific, but otherwise relatively nonselective, ion channel (Kelley et al. 2003). The MX helix on the cytoplasmic side of the membrane can modulate the function of the receptor, and its interactions with membrane lipids play a major role (Mocatta et al. 2022).

5HT3B receptor of Homo sapiens

Zinc-activated ligand-gated ion channel isoform X1, ZACN-X1, of 662 aas and 11 TMSs in a 4 + 1 + 3 + 1 TMS arrangement.  The first 4 TMSs (about residues 1 - 244) are homologous to residues 153 - 398 in the opioid receptor (TC# 9.A.14.13.18) while most of the rest of the protein is homologous to TC# 1.A.9.2.4 (residues ~191 - 662). This latter region shows a 3 + 1 TMS arrangement as is true for most members of the LIC family proteins.

ZACN-X1 of Odocoileus virginianus texanus

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). Residues involved in glucose sensitivity of recombinant human glycine receptors have been identified (Hussein et al. 2020). Lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor's conformational dynamics ((Dämgen and Biggin 2021)). Some protein-lipid interactions occur at a site at the communication interface between the extracellular and transmembrane domain, and in the active state, cholesterol can bind to the binding site of the positive allosteric modulator, ivermectin (Dämgen and Biggin 2021). An intracellular domain determines the agonist specificity (Ivica et al. 2021). The general anesthetic etomidate and fenamate mefenamic acid oppositely affect GABAAR and GlyR. These drugs potentiated GABAARs but blocked GlyRs (Rossokhin 2020). Alpha 1 glycine receptors are strongly inhibited by two flavanoids, quercetin and naringenin (Breitinger et al. 2021). The glycine receptor beta-subunit A455P variant occurs in a family affected by hyperekplexia syndrome (Aboheimed et al. 2022). Evidence for distinct roles of conserved proline residues in GlyR has been presented (Lummis and Dougherty 2022). Cannabinoids in general, and THC in particular, modulate pain perception via GlyR with possible clinical applications (Alvarez and Alves 2022). A set of functionally essential but differentially charged amino-acid residues in the transmembrane domain of the alpha1 and beta subunits explains asymmetric activation. These findings point to a gating mechanism that is distinct from homomeric receptors but more compatible with heteromeric GlyRs, being clustered at synapses through beta subunit-scaffolding protein interactions (Liu and Wang 2023). Such a mechanism provides a foundation for understanding how gating of the Cys-loop receptor members diverge to accommodate a specific physiological environment. Gallagher et al. 2022 reviewed the structural basis for how current compounds cause positive allosteric modulation of glycine receptors and discusses their therapeutic potential as analgesics.

Animals

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).

anthropods

HclA of Drosophila melanogaster (A1KYB4)

Glutamate receptor of 552 aas, GluCl-2 (Lynagh et al. 2014).

Animals

GluCl-2 of Schistosoma mansoni (Blood fluke)

Lgc-40 of Caenorhabditis elegans (Q22741)

Glycine receptor, GlyR or GlyREM with two subunits, Glra4a (subunit GlyRα; 459 aas) and Glra4b (subunit Glrb or GlyRβ; 498 aas). These subunits are about 80% identical to the human subunits (TC# 1.A.9.3.1). Agonists include glycine, β-alanine, GABA and taurine (Ivica et al. 2021). Intracellular loop domains (ICD) in part determine the agonist specificity and efficiency (Ivica et al. 2021). Lateral fenestrations between subunits in the extracellular domain provide the main translocation pathway for chloride ions to enter/exit a central water-filled vestibule at the entrance of the transmembrane channel (Cerdan et al. 2022).

GlyRα/GlyRβ of Danio rerio (Zebrafish) (Brachydanio rerio)

Histamine-gated chloride channel 2 (HACl2) of 425 aas and 4 TMSs. HACls mediate fast inhibitory neurotransmission in invertebrate nervous systems and have important roles in light reception, color processing, temperature preference and light-dark cycles. The fall armyworm, Spodoptera frugiperda is a primary destructive pest of grain and row crops (Yin et al. 2021). Histamine (HA) and gamma-aminobutyric acid (GABA) activated inward currents when SfHACls were singly or collectively expressed with different ratios in Xenopus laevis oocytes. These channels were ~2000-fold more sensitive to HA than to GABA. They were anion-selective channels that were highly dependent on changes in external chloride concentrations, but insensitive to changes in external sodium concentrations. The insecticides abamectin (ABM) and emamectin benzoate (EB) also activated these channels (Yin et al. 2021).

HACl of Spodoptera frugiperda (fall armyworm)

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). Fipronil is a GABA-gated chloride channel blocker (Pfaff et al. 2021). 

Animals

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). A database of glutamate-gated chloride (GluCl) subunits across 125 nematode species reveals patterns of gene accretion and sequence diversification (O'Halloran 2022).

Animals

GluCl of Caenorhabditis elegans
Avr-14 (Q8IFY7)
Avr-15 (Q9TW41)
Glc-1 (O17793)

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).

Animals

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).

Animals

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, GABARA1 or GABAAR. 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). Many (but not all) sedative-hypnotics are capable of positively modulating the GABAA receptor by binding within a common set of hydrophobic cavities (McGrath et al. 2020). Isoflurane binds to a site within the transmembrane domains of the receptor and suggest functional similarity between the GABA(A) alpha-1, -2, and -3 subunits (Schofield and Harrison 2005). Mutations ain the M2 and M3 TMSs of the GABAARs alpha1 and beta2 subunits affect late gating transitions including opening/closing and desensitization (Terejko et al. 2021). The distance between an alpha1beta3gamma2L GABA type A receptor residue and the drug, etomidate, when bound in the transmembrane beta+/alpha- interface, has been determined (Fantasia et al. 2021). There is a binding site in the beta(+)alpha(-) interface for the anesthetic, propofol (Borghese et al. 2021). Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is widely used to study selective actions mediated by delta-subunit-containing GABAA receptors. The molecular determinants responsible for positive modulation by DS2 have been identified (Falk-Petersen et al. 2021). Two high-resolution structures of GABAA receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the alpha/gamma subunit interface and two transmembrane sites at beta/alpha interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success (Zhu et al. 2022). Molecular dynamics simulations provided insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy (Zhu et al. 2022). Avermectin-imidazo[1,2-a]pyridine hybrids are potent GABAA receptor modulators (Volkova et al. 2022). Clptm1 is a target for suppressing epileptic seizures by regulating GABA(A) R-mediated inhibitory synaptic transmission in a PTZ-induced epilepsy model (Zhang et al. 2023). The allosteric modulation of α1β3γ2 GABA(A) receptors by farnesol through neurosteroid sites has been characterized (Gc et al. 2023). Chloride ion dysregulation in epileptogenic neuronal networks has been reviewed (Weiss 2023).

Animals

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)

The Rdl (Rd1) GABA receptor of 606 aas and 4 TMSs. Amino acids important for function have been identified (Nakao and Banba 2020).

Rd1 GABA receptor of Drosophila melanogaster

γ-aminobutyric acid (GABA) receptor subunit beta of 499 aas and 4 TMSs, one N-terminal, one C-terminal, and two plus a central P-loop, near the C-terminal end of the protein. GABA, an inhibitory neurotransmitter, mediates neuronal inhibition by binding to the GABA/benzodiazepine receptor and opening an integral chloride channel. PNU-120596, a positive allosteric modulator of mammalian alpha7 nicotinic acetylcholine receptor, is a negative modulator of ligand-gated chloride-selective channels of the gastropod, Lymnaea stagnalis (Vulfius et al. 2020).

GABA recptor of Lymnaea stagnalis (Great pond snail) (Helix stagnalis)

GABAAR beta subunit of 474 aas and 4 TMSs in a 3 + 1 TMS arrangement. γ-aminobutyric acid type A receptors (GABAARs) are ligand gated channels mediating inhibition in the central nervous system. Garifulina et al. 2022 identified a previouly undescribed function of beta-subunit homomers as proton-gated anion channels. Mutation of a single H267A in beta3 subunits completely abolished channel activation by protons. In molecular dynamic simulations of the beta3 crystal structure, protonation of H267 increased the formation of hydrogen bonds between H267 and E270 of the adjacent subunit, leading to a pore stabilising ring formation and accumulation of Cl^- within the transmembrane pore. Conversion of these residues in proton insensitive rho1 subunits transfered proton-dependent gating, thus highlighting the role of this interaction in proton sensitivity. Activation of chloride and bicarbonate currents at physiological pH, and kinetic studies, suggest a physiological role in neuronal and non-neuronal tissues that express beta subunits, and thus they are potential novel drug target (Garifulina et al. 2022). Residues in the 1st TMS are important for GABAArho receptor function (Crowther et al. 2022).

Beta subunit of Homo sapiens

Gamma-aminobutyric acid receptor subunit alpha-5, GABRA5, of 462 aas and 4 TMSs in a 3 + 1 C-terminal arrangement. It is a ligand-gated chloride channel subunit which is a component of the heteropentameric receptor for GABA, the major inhibitory neurotransmitter in the brain (Butler et al. 2018, Hernandez et al. 2019). It may be involved in GABA-A receptor assembly, and GABA-A receptor immobilization and accumulation by gephyrin at the synapse (Hernandez et al. 2019). Ablation of Gabra5 influences corticosterone levels and anxiety-like behavior in mice (Syding et al. 2023).

GABRA5 of Homo sapiens

γ-Aminobutyric acid (GABA)-inhibited Cl^- channel, type A (α-, β- γ-subunit precursors), GABRA2 or GABAAR2, regulated by GABA receptor accessory protein, GABARAP (Luu et al., 2006) and FRMD7 (TC# 8.A.25.1.5) (Jiang et al. 2020). A mutation in the GABAA receptor alpha 1 subunit, linked to human epilepsy, affects channel gating properties (Fisher 2004). 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). Elevin novel molecules, identified using reinforcement learning, showed positive allosteric modulation, with two showing 50% activation in the low micromolar range (Michaeli et al. 2020). GABAA Receptor ligands interact with binding sites in the transmembrane domain and in the extracellular domain (Iorio et al. 2020). Many (but not all) sedative-hypnotics are capable of positively modulating the GABAA receptor by binding within a common set of hydrophobic cavities (McGrath et al. 2020). Allopregnanolone (3alpha5alpha-P), pregnanolone), and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Photoaffinity labeling procedures have been used to identify an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors (Jayakar et al. 2020). Diazepam binds to etomidate binding sites in the transmembrane receptor domain giving rise to antagonism (McGrath et al. 2020). The alpha1 subunit histidine 55 at the interface between the extracellular and transmembrane domains affects preactivation and desensitization of the GABAA receptor (Kaczor et al. 2021). Coordinated downregulation of KCC2 and the GABAA receptor contributes to inhibitory dysfunction during seizure induction (Wan et al. 2020). Loss of GABAergic inhibition provides a mechanism underlying GABRB2-associated neurodevelopmental disorders (El Achkar et al. 2021). GABAAR binds the anaesthetic, Propofol, to induced conformational changes (Yuan et al. 2021). Methaqualone (2-methyl-3-(o-tolyl)-quinazolin-4(3H)-one, MTQ) is a moderately potent positive allosteric modulator (PAM) of GABAA receptors (GABAARs). Several additional potent GABAAR PAMs  include 2,3-diphenylquinazolin-4(3H)-one (PPQ), 3-(2-chlorophenyl)-2-phenylquinazolin-4(3H)-one (Cl-PPQ), and others (Wang et al. 2020). Interfacial binding sites for cholesterol on GABAA receptors compete with neurosteroids (Lee 2021). GABAAR is inhibited by L-type calcium channel blockers (Das et al. 2004). In in vivo studies, Stigmasterol (0.5-3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner to allopregnanolone, indicating the involvement of a GABAergic mechanism. Thus, GABAA receptors are subject to anxiolytic and anticonvulsant activities of stigmasterol. Thus, stigmasterol is a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors (Karim et al. 2021). Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric properties that affect inhibitoryGABAA receptors (Janzen et al. 2021). High-dose benzodiazepines positively modulate GABAA receptors via a flumazenil-insensitive mechanism (Wang et al. 2021). Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high anaesthetic concentrations (McGrath et al. 2021). (+)-Catharanthine potentiates the GABAA receptor by binding to a transmembrane site at the beta(+)/alpha(-) interface near the TMS2-TMS3 loop (Arias et al. 2022). Diazepam derivatives are allosteric modulators of GABAA receptor alpha1beta2gamma2 subtypes (Djebaili et al. 2022). α1 proline 277 residues regulate GABAAR gating through M2-M3 loop interactions in the interfacial region (Kaczor et al. 2022). Regulated assembly and neurosteroid modulation constrain GABA(A) receptor pharmacology in vivo (Sun et al. 2023).

Animals

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)
GABARAP (O95166)

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). Isocycloseram is a "GABA-gated chloride channel allosteric modulator (Blythe et al. 2022).

Animals (insects)

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). A variant of GABRA1 (A332V) causes increased sensitivity for GABA and alterred desensitization (Steudle et al. 2020). Pathogenic variants in GABRB3 have been associated with a spectrum of phenotypes from severe developmental disorders and epileptic encephalopathies to milder epilepsy syndromes and mild intellectual disability (Johannesen et al. 2021).

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). Isocycloseram is a "GABA-gated chloride channel allosteric modulator (Blythe et al. 2022).

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. Neuroligin 3 from the common cutworm enhances the GABA-induced current of a recombinant SlRDL1 channel (Wang et al. 2021). GABA receptor channels are blocked by the ectoparasiticide, fluralaner (Kono et al. 2022).

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). Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is widely used to study selective actions mediated by delta-subunit-containing GABAA receptors. The molecular determinants responsible for positive modulation by DS2 have been identified (Falk-Petersen et al. 2021).

GabaA1 receptor of Danio rerio (Zebrafish)

Animals

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).

Animals

Lgc-55 of Caenorhabditis elegans (Q9TVI7)

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 revealed 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 demonstrated 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). Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction. Lycksell et al. 2021 used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of GLIC under resting and activating conditions. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Thus, the findings demonstrate state-dependent changes in a pentameric ion channel by SANS. A 3-state  model has been proposed; mutations at the subunit interface in the extracellular domain (ECD) principally alter pre-activation, while mutations in the lower ECD and the transmembrane domain principally alter activation. Propofol alters both transitions (Lefebvre et al. 2021). Cryo-EM structures of GLIC under three pH conditions showed that decreased pH is associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the beta1-beta2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore (Rovšnik et al. 2021). Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) by selectively binding to a single site in the outer transmembrane domain of ELIC (Dietzen et al. 2022).

Bacteria

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).

Proteobacteria

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. The channel is regulated by a semi-conserved cationic-lipid binding site, where the residue involved is the tryptophan, W206 (Sridhar et al. 2021).

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). Ligand-binding affinities are insensitive to binding-site occupancy, and mutations in residues in the transmembrane domain are unlikely to affect the channel's affinities for ligands that bind to the extracellular domain (Godellas and Grosman 2022).

CLIC or pLGIC of Desulfofustis deltaproteobacterium