The Concise Guide to Pharmacology 2013/14: Ligand-Gated Ion Channels

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. Ligand-gated ion channels are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates.


An Introduction to Ligand-gated Ion Channels
Ligand-gated ion channels (LGICs) are integral membrane proteins that contain a pore which allows the regulated flow of selected ions across the plasma membrane. Ion flux is passive and driven by the electrochemical gradient for the permeant ions. The channels are opened, or gated, by the binding of a neurotransmitter to an orthosteric site(s) that triggers a conformational change that results in the conducting state. Modulation of gating can occur by the binding of endogenous, or exogenous, modulators to allosteric sites.
LGICs mediate fast synaptic transmission, on a millisecond time scale, in the nervous system and at the somatic neuromuscular junction. Such transmission involves the release of a neurotransmitter from a pre-synaptic neurone and the subsequent activation of post-synaptically located receptors that mediate a rapid, phasic, electrical signal (the excitatory, or inhibitory, post-synaptic potential). However, In addition to their traditional role in phasic neurotransmission, it is now established that some LGICs mediate a tonic form of neuronal regulation that results from the activation of extrasynaptic receptors by ambient levels of neurotransmitter. The expression of some LGICs by non-excitable cells is suggestive of additional functions.
By convention, the LGICs comprise the excitatory, cationselective, nicotinic acetylcholine Changeux, 2010), 5-HT3 ( Barnes et al., 2009;, ionotropic glutamate Traynelis et al., 2010) and P2X receptors (Jarvis and Khakh, 2009;Surprenant and North, 2009) and the inhibitory, anion-selective, GABAA Belelli et al., 2009) and glycine receptors (Lynch, 2009;Yevenes and Zeihofer, 2011). The nicotinic acetylcholine, 5-HT3, GABAA and glycine receptors (and an additional zincactivated channel) are pentameric structures and are frequently referred to as the Cys-loop receptors due to the presence of a defining loop of residues formed by a disulphide bond in the extracellular domain of their constituent subunits (Miller and Smart, 2010; Thompson et al., 2010). However, the prokaryotic ancestors of these receptors contain no such loop and the term pentameric ligand-gated ion channel (pLGIC) is gaining acceptance in the literature ( glutamate and P2X receptors are tetrameric and trimeric structures, respectively. Multiple genes encode the subunits of LGICs and the majority of these receptors are heteromultimers. Such combinational diversity results, within each class of LGIC, in a wide range of receptors with differing pharmacological and biophysical properties and varying patterns of expression within the nervous system and other tissues. The LGICs thus present attractive targets for new therapeutic agents with improved discrimi-nation between receptor isoforms and a reduced propensity for off-target effects. The development of novel, faster screening techniques for compounds acting on LGICs (Dunlop et al., 2008) will greatly aid in the development of such agents.

Channels
Notably, most ligands display significantly reduced affinities at the guinea-pig 5-HT3 receptor in comparison with other species.
In addition to the agents listed in the table, native and recombinant 5-HT3 receptors are subject to allosteric modulation by extracellular divalent cations, alcohols, several general anaesthetics and 5-hydroxy-and halide-substituted indoles (see reviews [36,[44][45]50]).

GABA A receptors
Overview: The GABAA receptor is a ligand-gated ion channel of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3 and strychnine-sensitive glycine receptors. GABAA receptor-mediated inhibition within the CNS occurs by fast synaptic transmission, sustained tonic inhibition and temporally intermediate events that have been termed 'GABAA, slow' [9]. GABAA receptors exist as pentamers of 4TM subunits that form an intrinsic anion selective channel. Sequences of six α, three β, three γ, one δ, three ρ, one ε, one π and one θ GABAA receptor subunits (gene family ID ENSF00000000053) have been reported in mammals [36][37]42,44]. The π-subunit is restricted to reproductive tissue. Alternatively spliced versions of many subunits exist (e.g. α4and α6-(both not functional) α5-, β2-, β3and γ2), along with RNA editing of the α3 subunit [12]. The three ρ-subunits, (ρ1-3) function as either homo-or hetero-oligomeric assemblies [10,55]. Receptors formed from ρ-subunits, because of their distinctive pharmacology that includes insensitivity to bicuculline, benzodiazepines and barbiturates, have sometimes been termed GABAC receptors [55], but they are classified as GABAA receptors by NC-IUPHAR on the basis of structural and functional criteria [3,[36][37].
Many GABAA receptor subtypes contain α-, βand γ-subunits with the likely stoichiometry 2α.2β.1γ [26,36]. It is thought that the majority of GABAA receptors harbour a single type of αand β -subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABAA receptors in the CNS, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that incorporate the α4-α5-or α6-subunit, or the β1-, γ1-, γ3-, δ-, εand θ-subunits, are less numerous, but they may nonetheless serve important functions. For example, extrasynaptically located receptors that contain α6and δ-subunits in cerebellar granule cells, or an α4and δ-subunit in dentate gyrus granule cells and thalamic neurones, mediate a tonic current that is important for neuronal excitability in response to ambient concentrations of GABA [4,13,32,40,45]. GABA binding occurs at the β+/αsubunit interface and the homologous γ+/αsubunits interface creates the benzodiazepine site. A second site for benzodiazepine binding has recently been postulated to occur at the α+/βinterface ( [38]; reviewed by [43]). The particular α-and γ-subunit isoforms exhibit marked effects on recognition and/or efficacy at the benzodiazepine site. Thus, receptors incorporating either α4or α6-subunits are not recognised by 'classical' benzodiazepines, such as flunitrazepam (but see [104]). The trafficking, cell surface expression, internalisation and function of GABAA receptors and their subunits are discussed in detail in several recent reviews [61,70,81,101] but one point worthy of note is that receptors incorporating the γ2 subunit (except when associated with α5) cluster at the postsynaptic membrane (but may distribute dynamically between synaptic and extrasynaptic locations), whereas as those incorporating the d subunit appear to be exclusively extrasynaptic.
NC-IUPHAR [53,86] class GABAA receptors according to their subunit structure, pharmacology and receptor function. Currently, eleven native GABAA receptors are classed as conclusively identified (i.e., α1β2γ2, α1βγ2, α3βγ2, α4βγ2, α4β2δ, α4β3δ, α5βγ2, α6βγ2, α6β2δ, α6β3δ and ρ) with further receptor isoforms occurring with high probability, or only tentatively [86][87]. It is beyond the scope of this Guide to discuss the pharmacology of individual GABAA receptor isoforms in detail; such information can be gleaned in the reviews [53,66,71,76,78,83,[86][87]92] and [51][52]. Agents that discriminate between α-subunit isoforms are noted in the table and additional agents that demonstrate selectivity between receptor isoforms, for example via β-subunit selectivity, are indicated in the text below. The distinctive agonist and antagonist pharmacology of ρ receptors is summarised in the table and additional aspects are reviewed in [60,72,84,105]. Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [77] Comments: isonipectoic acid is a relatively high efficacy agonist at the GABA binding site of α4 and α6 subunits. Diazepam and flunitrazepam are not active at α4or α6-subunits. [ 11 C]flumazenil is a low affinity ligand at the benzodiazepine site of α4 and α6 subunits. [ 3 H]Ro154513 selectively labels α4and α6-subunit containing receptors in the presence of a saturating concentration of a 'classical' benzodiazepine (e.g. diazepam).

Subunits
Channel Blockers picrotoxin, TBPS Comment Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively [77] Nomenclature δ ε θ π Zn 2+ is an endogenous allosteric regulator and causes potent inhibition of receptors formed from binary combinations of α and β subunits, incorporation of a δ or γ subunit causes a modest, or pronounced, reduction in inhibitory potency, respectively Nomenclature ρ1 ρ2 ρ3 The potency and efficacy of many GABA agonists vary between receptor GABAA receptor isoforms [66,73,78]. For example, THIP (gaboxadol) is a partial agonist at receptors with the subunit composition α4β3γ2, but elicits currents in excess of those evoked by GABA at the α4β3δ receptor where GABA itself is a low efficacy agonist [56,58]. The antagonists bicuculline and gabazine differ in their ability to suppress spontaneous openings of the GABAA receptor, the former being more effective [98]. The presence of the γ subunit within the heterotrimeric complex reduces the potency and efficacy of agonists [96]. The GABAA receptor contains distinct allosteric sites that bind barbiturates and endogenous (e.g., 5α-pregnan-3α-ol-20-one) and synthetic (e.g., alphaxalone) neuroactive steroids in a diastereo-or enantio-selective manner [55,[68][69]100]. Picrotoxinin and TBPS act at an allosteric site within the chloride channel pore to negatively regulate channel activity; negative allosteric regulation by γ-butyrolactone derivatives also involves the picrotoxinin site, whereas positive allosteric regulation by such compounds is proposed to occur at a distinct locus. Many intravenous (e.g., etomidate, propofol) and inhalational (e.g., halothane, isoflurane) anaesthetics and alcohols also exert a regulatory influence upon GABAA receptor activity [57,85]. Specific amino acid residues within GABAA receptor αand β-subunits that influence allosteric regulation by anaesthetic and non-anaesthetic compounds have been identified [67,69]. Photoaffinity labelling of distinct amino acid residues within purified GABAA receptors by the etomidate derivative, [ 3 H]azietomidate, has also been demonstrated [80] and this binding subject to positive allosteric regulation by anaesthetic steroids [79]. An array of natural products including flavonoid and terpenoid compounds exert varied actions at GABAA receptors (reviewed in detail in [71]).

Glycine receptors
Overview: The inhibitory glycine receptor [nomenclature as agreed by the NC-IUPHAR sub-committee on glycine receptors] is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors [121]. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [107,111], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1 INS and α1 del ), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [109,124,128]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the mature form of the receptor contains α1 (or α3) and β subunits while the immature form is mostly composed of only α2 subunits. RNA transcripts encoding the α4-subunit have not been detected in adult humans. The N-terminal domain of the α-subunit contains both the agonist and strychnine binding sites that consist of several discontinuous regions of amino acids. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracel-lular loop region, to gephyrin. The latter is a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [116][117]126]. G-protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [135][136]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [129]. Intracellular Ca 2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [110].

Subunits
Nomenclature α1 α2 α3  Comments: Data in the table refer to homo-oligomeric assemblies of the α-subunit, significant changes introduced by co-expression of the β1 subunit are indicated in parenthesis. Not all glycine receptor ligands are listed within the table, but some that may be useful in distinguishing between glycine receptor isoforms are indicated (see detailed view pages for each subunit: α1, α2, α3, α4, β). pregnenolone sulphate, tropisetron and colchicine, for example, although not selective antagonists of glycine receptors, are included for this purpose. strychnine is a potent and selective competitive glycine receptor antagonist with affinities in the range 5-15 nM. RU5135 demonstrates comparable potency, but additionally blocks GABAA receptors. There are conflicting reports concerning the ability of cannabinoids to inhibit [119], or potentiate and at high concentrations activate [106,108,112,[131][132] glycine receptors. Nonetheless, cannabinoid analogues may hold promise in distinguishing between glycine receptor subtypes [132]. In addition, potentiation of glycine receptor activity by cannabinoids has been claimed to contribute to cannabis-induced analgesia relying on Ser296/307 (α1/α3) in M3 [131]. Several analogues of muscimol and piperidine act as agonists and antagonists of both glycine and GABAA receptors. picrotoxin acts as an allosteric inhibitor that appears to bind within the pore, and shows strong selectivity towards homomeric receptors. While its components, picrotoxinin and picrotin, have equal potencies at α1 receptors, their potencies at α2 and α3 receptors differ modestly and may allow some distinction between different receptor types [133]. Binding of picrotoxin within the pore has been demonstrated in the crystal structure of the related C. elegans GluCl Cys-loop receptor [113]. In addition to the compounds listed in the table, numerous agents act as allosteric regulators of glycine receptors (comprehensively reviewed in [118,120,130,137]). Zn 2+ acts through distinct binding sites of high-and low-affinity to allosterically enhance channel function at low (<10 μM) concentrations and inhibits responses at higher concentrations in a subunit selective manner [125]. The effect of Zn 2+ is somewhat mimicked by Ni 2+ . Endogenous Zn 2+ is essential for normal glycinergic neurotransmission mediated by α1 subunit-containing receptors [114]. Elevation of intracellular Ca 2+ produces fast potentiation of glycine receptor-mediated responses. Dideoxyforskolin (4 μM) and tamoxifen (0.2-5 μM) both potentiate responses to low glycine concentrations (15 μM), but act as inhibitors at higher glycine concentrations (100 μM). Additional modulatory agents that enhance glycine receptor function include inhalational, and several intravenous general anaesthetics (e.g. minaxolone, propofol and pentobarbitone) and certain neurosteroids. ethanol and higher order n-alcohols also enhance glycine receptor function although whether this occurs by a direct allosteric action at the receptor [123], or through G protein βγ subunits [134] is debated. Recent crystal structures of the bacterial homologue, GLIC, have identified transmembrane binding pockets for both anaesthetics [127] and alcohols [115]. Solvents inhaled as drugs of abuse (e.g. toluene, 1-1-1-trichloroethane) may act at sites that overlap with those recognising alcohols and volatile anaesthetics to produce potentiation of glycine receptor function. The function of glycine receptors formed as homomeric complexes of α1 or α2 subunits, or hetero-oligomers of α1/β or α2/β subunits, is differentially affected by the 5-HT3 receptor antagonist tropisetron (ICS 205-930) which may evoke potentiation (which may occur within the femtomolar range at the homomeric glycine α1 receptor), or inhibition, depending upon the subunit composition of the receptor and the concentrations of the modulator and glycine employed. Potentiation and inhibition by tropeines involves different binding modes [122]. Additional tropeines, including atropine, modulate glycine receptor activity.

Ionotropic glutamate receptors
Overview: The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [149,174,197]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), three transmembrane domains composed of three membrane spans (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy-terminal domain (CTD) [166,169,177,181,197]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2 -see below) has recently been solved at 3.6Å resolution [194] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [167]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [144,[148][149]153,[164][165]168,[184][185][186][197][198]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.
The classification of glutamate receptor subunits has been recently been re-addressed by NC-IUPHAR [146]. The scheme developed recommends a revised nomenclature for ionotropic glutamate receptor subunits that is adopted here.
AMPA and Kainate receptors AMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [154,163,179,195]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [171,[188][189]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [165,[188][189]). Kainate receptors may also exhibit 'metabotropic' functions [171,191]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [172,188]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Clfor their activation [141,190]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca 2+ ; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and Joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [162,192]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [171,188]. Native AMPA and kainate receptors displaying differential channel conductances, Ca 2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [147, 162,173]. GluA1-4 can exist as two variants generated by alternative splicing (termed 'flip' and 'flop') that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [179]. Splice variants of GluK1-3 also exist which affects their trafficking [171,188].  [163,179]. AMPA is weak partial agonist at GluK1 and at heteromeric assemblies of GluK1/GluK2, GluK1/ GluK5 and GluK2/GluK5 [165]. Quinoxalinediones such as CNQX and NBQX show limited selectivity between AMPA and kainate receptors. LY293558 also has kainate (GluK1) receptor activity as has GYKI53655 (GluK3 and GluK2/GluK3) [165]. ATPO is a potent competitive antagonist of AMPA receptors, has a weaker antagonist action at kainate receptors comprising GluK1 subunits, but is devoid of activity at kainate receptors formed from GluK2 or GluK2/GluK5 subunits. The pharmacological activity of ATPO resides with the (S)-enantiomer. ACET and UBP310 may block GluK3, in addition to GluK1 [138,187]. (2S,4R)-4-methylglutamate (SYM2081) is equipotent in activating (and desensitising) GluK1 and GluK2 receptor isoforms and, via the induction of desensitisation at low concentrations, has been used as a functional antagonist of kainate receptors. Both (2S,4R)-4-methylglutamate and LY339434 have agonist activity at NMDA receptors. (2S,4R)-4-methylglutamate is also an inhibitor of the glutamate transporters EAAT1 and EAAT2.

Subunits
Delta subunits GluD1 (GRID1, Q9ULK0) and GluD2 (GRID2, O43424) comprise, on the basis of sequence homology, an 'orphan' class of ionotropic glutamate receptor subunit. They do not form a functional receptor when expressed solely, or in combination with other ionotropic glutamate receptor subunits, in transfected cells [199]. However, GluD2 subunits bind D-serine and glycine and GluD2 subunits carrying the mutation A654T form a spontaneously open channel that is closed by D-serine [182].
NMDA receptors NMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [145,152]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [157,167,177]. However, more complex tri-heteromeric assem-blies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca 2+ and is blocked, in a voltage-dependent manner, by Mg 2+ such that at resting potentials the response is substantially inhibited. Channel Blockers -  Channel Blockers  [144,150,153,170,186,197]. In addition to the glutamate and glycine binding sites documented in the table, physiologically important inhibitory modulatory sites exist for Mg 2+ , Zn 2+ , and protons [148- 149,197]. Voltage-independent inhibition by Zn 2+ binding with high affinity within the ATD is highly subunit selective (GluN2A >> GluN2B > GluN2C ≥ GluN2D; [186,197]). The receptor is also allosterically modulated, in both positive and negative directions, by endogenous neuroactive steroids in a subunit dependent manner [161,176]. Tonic proton blockade of NMDA receptor function is alleviated by polyamines and the inclusion of exon 5 within GluN1 subunit splice variants, whereas the non-competitive antagonists ifenprodil and CP101606 (traxoprodil) increase the fraction of receptors blocked by protons at ambient concentration. Inclusion of exon 5 also abolishes potentiation by polyamines and inhibition by Zn 2+ that occurs through binding in the ATD [196]. Ifenprodil, CP101606, haloperidol, felbamate and Ro8-4304 discriminate between recombinant NMDA receptors assembled from GluN1 and either GluN2A, or GluN2B, subunits by acting as selective, noncompetitive, antagonists of heterooligomers incorporating GluN2B through a binding site at the ATD GluN1/GluN2B subunit interface [167]. LY233536 is a competitive antagonist that also displays selectivity for GluN2B over GluN2A subunitcontaining receptors. Similarly, CGP61594 is a photoaffinity label that interacts selectively with receptors incorporating GluN2B versus GluN2A, GluN2D and, to a lesser extent, GluN2C subunits. TCN 201 and TCN 213 have recently been shown to block GluN2A NMDA receptors selectively by a mechanism that involves allosteric inhibition of glycine binding to the GluN1 site [140,151,159,178]. In addition to influencing the pharmacological profile of the NMDA receptor, the identity of the GluN2 subunit co-assembled with GluN1 is an important determinant of biophysical properties that include sensitivity to block by Mg 2+ , single-channel conductance and maximal open probablity and channel deactivation time [148,152,158]. Incorporation of the GluN3A subunit into tri-heteromers containing GluN1 and GluN2 subunits is associated with decreased single-channel conductance, reduced permeability to Ca 2+ and decreased susceptibility to block by Mg 2+ [142,160]. Reduced permeability to Ca 2+ has also been observed following the inclusion of GluN3B in tri-heteromers. The expression of GluN3A (GRIN3A, Q8TCUS), or GluN3B (GRIN3B, O60391), with GluN1 alone forms, in Xenopus laevis oocytes, a cation channel with unique properties that include activation by glycine (but not NMDA), lack of permeation by Ca 2+ and resistance to blockade by Mg 2+ and NMDA receptor antagonists [143]. The function of heteromers composed of GluN1 and GluN3A is enhanced by Zn 2+ , or glycine site antagonists, binding to the GluN1 subunit [175]. Zn 2+ also directly activates such complexes. The co-expression of GluN1, GluN3A and GluN3B appears to be required to form glycineactivated receptors in mammalian cell hosts [193].

Nicotinic acetylcholine receptors
Overview: Nicotinic acetylcholine receptors are members of the Cys-loop family of transmitter-gated ion channels that includes the GABAA, strychnine-sensitive glycine and 5-HT3 receptors [201,229,[235][236]241]. All nicotinic receptors are pentamers in which each of the five subunits contains four α-helical transmembrane domains. Genes (Ensembl family ID ENSF00000000049) encoding a total of 17 subunits (α1-10, β1-4, γ, δ and ε) have been identified [224]. All subunits with the exception of α8 (present in avian species) have been identified in mammals. All α subunits possess two tandem cysteine residues near to the site involved in acetylcholine binding, and subunits not named α lack these residues [229]. The orthosteric ligand binding site is formed by residues within at least three peptide domains on the α subunit (principal component), and three on the adjacent subunit (complementary component). nAChRs contain several allosteric modulatory sites. One such site, for positive allosteric modulators (PAMs) and allosteric agonists, has been proposed to reside within an intrasubunit cavity between the four transmembrane domains [215,243]; see also [220]). The high resolution crystal structure of the molluscan acetylcholine binding protein, a structural homologue of the extracellular binding domain of a nicotinic receptor pentamer, in complex with several nicotinic receptor ligands (e.g. [208]) and the crystal structure of the extracellular domain of the α1 subunit bound to α-bungarotoxin at 1.94 Å resolution [213], has revealed the orthosteric binding site in detail (reviewed in [209,224,[234][235]). Nicotinic receptors at the somatic neuromuscular junction of adult animals have the stoichiometry (α1)2β1δε, whereas an extrajunctional (α1)2β1γδ receptor predominates in embryonic and denervated skeletal muscle and other pathological states.
Other nicotinic receptors are assembled as combinations of α(2-6) and &beta (2)(3)(4) subunits. For α2, α3, α4 and β2 and β4 subunits, pairwise combinations of α and β (e.g. α3β4 and α4β2) are sufficient to form a functional receptor in vitro, but far more complex isoforms may exist in vivo (reviewed in [217][218]229]). There is strong evidence that the pairwise assembly of some α and β subunits can occur with variable stoichiometry [e.g. (α4)2(β2)2 or (α4)3(β2)2] which influences the biophysical and pharmacological properties of the receptor [229]. α5 and β3 subunits lack function when expressed alone, or pairwise, but participate in the formation of functional hetero-oligomeric receptors when expressed as a third subunit with another α and β pair [e.g. α4α5αβ2, α4αβ2β3, α5α6β2, see [229] for further examples]. The α6 subunit can form a functional receptor when co-expressed with β4 in vitro, but more efficient expression ensues from incorporation of a third partner, such as β3 [242]. The α7, α8, and α9 subunits form functional homo-oligomers, but can also combine with a second subunit to constitute a hetero-oligomeric assembly (e.g. α7β2 and α9α10). For functional expression of the α10 subunit, co-assembly with α9 is necessary. The latter, along with the α10 subunit, appears to be largely confined to cochlear and vestibular hair cells. Comprehensive listings of nicotinic receptor subunit combinations identified from recombinant expression systems, or in vivo, are given in [229]. In addition, numerous proteins interact with nicotinic ACh receptors modifying their assembly, trafficking to and from the cell surface, and activation by ACh (reviewed by [203,223,228]).
The nicotinic receptor subcommittee of NC-IUPHAR has recommended a nomenclature and classification scheme for nicotinic acetylcholine (nACh) receptors based on the subunit composition of known, naturally-and/or heterologously-expressed nACh receptor subtypes [226]. Headings for this table reflect abbreviations designating nACh receptor subtypes based on the predominant α subunit contained in that receptor subtype. An asterisk following the indicated α subunit denotes that other subunits are known to, or may, assemble with the indicated α subunit to form the designated nACh receptor subtype(s). Where subunit stoichiometries within a specific nACh receptor subtype are known, numbers of a particular subunit larger than 1 are indicated by a subscript following the subunit (enclosed in parentheses -see also [212]).

Selective channel blockers (IC50)
A-867744 (α4β2) [227], NS1738 (α4β2) [ Functional characteristics -(α9)5: PCa/PNa = 9; α9α10: PCa/PNa = 9, Pf = 22% Comments: Commonly used agonists of nACh receptors that display limited discrimination in functional assays between receptor subtypes include A-85380, cytisine, DMPP, epibatidine, nicotine and the natural transmitter, acetylcholine (ACh). A summary of their profile across differing receptors is provided in [218] and quantitative data across numerous assay systems are summarized in [222]. Quantitative data presented in the table for commonly used antagonists and channel blockers for human receptors studied under voltage-clamp are from [207,210,[230][231]233,240]. Type I PAMs increase peak agonist-evoked responses but have little, or no, effect on the rate of desensitization of α7 nicotinic ACh receptors whereas type II PAMs also cause a large reduction in desensitization (reviewed in [238]).

Subunits
Agonists listed show selectivity within recombinant P2X receptors of ca. one order of magnitude. A804598, A839977, A740003 and A438079 are at least 10-fold selective for P2X7 receptors and show similar affinity across human and rodent receptors [248,250,254].

Overview:
The zinc-activated channel [ZAC, nomenclature as agreed by the NC-IUPHAR Subcommittee for the zinc activated channel] is a member of the Cys-loop family that includes the nicotinic acetylcholine, 5-HT3, GABAA and strychnine-sensitive glycine receptors [278][279]. The channel is likely to exist as a homopentamer of 4TM subunits that form an intrinsic cation selective channel displaying constitutive activity that can be blocked by (+)-tubocurarine. ZAC is present in the human, chimpanzee, dog, cow and opossum genomes, but is functionally absent from mouse, or rat, genomes [278][279]. (+)-tubocurarine (6.3x10 -6 M) [278] Outwardly rectifying current (both constitutive and evoked by Zn 2+ )

Subunits
Although tabulated as an antagonist, it is possible that (+)-tubocurarine acts as a channel blocker