1.A.9 The Neurotransmitter Receptor, Cys loop, Ligand-gated Ion Channel (LIC) Family

Members of the LIC family of ionotropic neurotransmitter receptors (also called the cys-loop ligand gated ion channel family because of the signature cysteine loop in the amino terminal domain) are found in vertebrate and invertebrate animals and prokaryotes (TC# 1.A.9.8.1; Bocquet et al., 2007; Sine and Engel, 2006). Because of this extracellular N-terminal ligand-binding domain, they exhibit receptor specificity for (1) acetylcholine (AcCh), (2) serotonin, (3) glycine, (4) glutamate and (5) γ-aminobutyric acid (GABA)in vertebrates. All of these receptor channels are probably hetero- or homopentameric. The best characterized are the nicotinic acetylcholine receptors which are pentameric channels of α2βγδ (immature muscle) nα2βγδ (mature muscle; see 1.A.9.1.1) (Witzemann et al., 1990; Wada et al., 1998;(Khiroug et al., 2002) subunit composition. All subunits are homologous and have four transmembrane α-helices, M1-M4. Zn²⁺-activated cation channels of vertebrates and glutamate/serotonin-activated anion channels and GABA-gated cation channels of invertebrates are also in this family (Chen et al., 2006). Ligand binding has been reported to open the channel by a cis-trans prolylisomerization event (Cymes et al., 2005; Lummis et al., 2005).

The 5-HT(3) and acetylcholine receptors (cationic ion channels) and the GABA(A) and glycine receptors (anionic ion channels) generally depolarize or hyperpolarize, respectively, the neuronal membrane. Within the amino-terminal extracellular region, all members of this family exhibit a similar architecture of ligand binding domains, and a number of key residues are completely conserved (Connolly, 2008). Zhu and Hummer (2009) have concluded that the conformational transition from open and closed states involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.

The three-dimensional structures of the protein complex in both the open and closed configurations have been solved (Miyazawa et al., 2003). The five subunits (each of 400-500 amino acyl residues in length) are arranged in a ring with their 'M2' transmembrane helical spanners lining the central channel. The five M2 segments come together in the middle of the membrane to form the channel gate, and the gate opens upon binding of acetylcholine to distant sites in the N-terminal domains of the two α-subunits. The M2 segment determines the anion versus cation selectivity (Menard et al., 2005). These general structural features are probably valid for all members of the family. The acetylcholine receptor subunit consists of two domains, the channel domain and the ligand-binding extracellular domain. The latter is homologous to a soluble protein, the acetylcholine binding protein (AChBP), the structure of which has been solved at high resolution (Brejc et al., 2001).

Pentameric ligand-gated ion channels of the Cys-loop family mediate fast chemo-electrical transduction. Bocquet et al., 2009 presented the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation (TC #1.A.9.9.1), shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analyses of GLIC and ELIC revealed, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation (Bocquet et al., 2009).

According to Miyazawa et al. (2003), the pore is shaped by the inner ring of 5 α-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 α-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighboring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart (Miyazawa et al., 2003). (Nury et al., 2010) have presented a microsecond molecular dynamics simulation of channel gating in a nicotinic receptor homologue.

Bouzat et al. (2004) have identified structural requirements for functionally coupling the AcCh binding domain to the pore-forming domain. At least three loops in the AcCh binding domain interact with the pore domain to trigger channel opening. Modeling suggests a network of interacting loops between the two domains mediate allosteric coupling (Bouzat et al., 2004). Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ('Phi blocks') move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening, is comprised of residues near the transmitter binding site, and the second is comprised of residues near the base of the extracellular domain (Purohit and Auerbach, 2007).

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. Single-molecule kinetic analysis has been used to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the α-subunit of the murine acetylcholine receptor (Purohit et al., 2007). αM2 move in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.

GABA interacts with three kinds of receptors, classes A, B and C. Classes A and C receptors are ligand-gated Cl^- channels while class B receptors activate other channels via G proteins. GABA binding to both Class A and C receptors opens the Cl^- channels, leading to increased membrane conductance. These two classes of receptors differ in their antagonist specificities and therefore are distinguished pharmacologically. These receptors consist of 6 types of subunits: α, β, γ, δ, ε, and π. There are 6 αs, 3 βs and 3 γs, and 1 each of the δ, ε and π subunits. Usually the pentamer consists of 2αs, 2 βs and 1 γ, each with 4 putative TMSs, a long N-terminus and a short C-terminus, both extracellular. These receptors possess binding sites for anti-epileptic drugs, sedatives and anesthetics. Evidence suggests that the TMS1-2 hairpin loops of the 5 subunits comprise the GABA receptor pore (Filippova et al., 2004).

γ-Aminobutyric acid type A (GABA_A) receptors consist of subunits whose assembly forms a neurotransmitter-gated anion channel. Subunits for this receptor constitute a large family whose members are classified according to primary structure as α, β, γ, and δ, ε, π, and ρ subunits. Recombinant expression studies of different α variants in combination with a β variant and the γ₂ subunit demonstrated that GABA_A receptors with distinct pharmacological properties are generated. A distinctive mark of these α_xβ_xγ₂ receptors is their ability to bind modulatory compounds such as benzodiazepines (BZs), which can modulate γ-aminobutyric acid (GABA)-gated channel activity at an allosteric site. The α variants determine the affinity of GABA_A receptor subtypes toward these modulatory compounds, and members of the γ-subunit class, in particular the γ₂ variant, are essential for the architecture of the BZ site. Furthermore, the γ subunits impart a large unitary conductance on GABA_A channels (Herb et al., 1992).

γ₂ and δ subunits share approximately 35% sequence identity with α and β subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The γ₂ subunit is the rat homologue of the human γ₂ subunit recently shown to be important for benzodiazepine pharmacology (Shivers et al., 1989). Functional GABA receptors mediating Cl^- uptake in a picrotoxin-sensitive process (α₅, β₁, γ₁) have been identified and shown to be functional in renal proximal tubular cells (Sarang et al., 2008).

A novel type of GABA receptor has been characterized (Beg and Jorgensen, 2003). It is an excitatory GABA gated cation channel (TC #1.A.9.7.1). This channel is the EXP-1 receptor of the nematode, C. elegans. It is as divergent in sequence from the A and C class anion selective GABA receptors as it is from the other ligand-gated ion channel proteins in TCDB. It therefore represents a new subfamily in the LIC family. Inhibitory glycine receptors (GlyR) mediate Cl- influx and bind strychnine with nanomolar affinity. They are present in various nervous tissues (spinal cord, brain stem, caudal brain and retina (Cascio, 2004). Reduced activities of mutants often results in channelopathies involving muscle tone regulation including human startle disease (hyperekplexia). There are multiple subunit subtypes in humans (α₁, α₂, α₃, α₄ and β subunits) (Cascio, 2004)). Alternative splicing also occurs. In adults, the most common pentameric form consists of α₁ and β subunits. The M2α-helix region peptide of α₁ GlyR in lipid vesicles forms chloride-conducting pores, and sympathetic M2-based peptides form Cl^- channels in cell membranes (Mitchell et al., 2000).

There are seven classes of serotonin (5 hydroxytryptamine (5HT) receptors, six of which are G-protein linked, plus one of which is a homo- or heteropentameric ligand gated non-specific cation channel (TC #1.A.9.2.1 and 2) of the LIC family (Reeves and Lummis, 2002). Anion selective serotonin receptors may be present in C. elegans (TC #1.A.9.6.1). As for other LIC family members, four transmembrane hydrophobic segments (TMSs) (M1-M4) are predicted by hydropathy analysis.

The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] mediates rapid excitatory responses in peripheral and central neurons by activating ligand-gated ion channels (5-HT₃ receptors). These receptors are expressed in a variety of peripheral ganglia, where they are thought to modulate responses to pain, and to control reflexes of the enteric and cardiovascular systems. In the central nervous system, 5-HT₃ receptors have been implicated in the control of emesis, and antagonists of 5-HT₃ receptors have found clinical use for suppression of the nausea that accompanies postoperative recovery and many cancer therapies. Most families of ligand-gated ion channels are composed of multiple subunit types that assemble in alternative combinations to achieve functional diversity (Hanna et al., 2000).

Recombinant expression of 5-HT_3A subunits alone yields functional 5-HT₃ receptors, but, heteromeric assemblies of the human 5-HT_3A and 5-HT_3B subunits more closely resemble native 5-HT₃ receptors of rodent ganglia with respect to their single-channel conductance and permeability to Ca²⁺ ions. Consequently, it is likely that rodent ganglia normally express heteromeric 5-HT₃ receptors. However, the 5-HT₃ receptors of different species display several distinctive properties, particularly with respect to their pharmacological profiles (Hanna et al., 2000).

The channel protein complexes of the LIC family preferentially transport cations or anions depending on the channel (e.g., the acetylcholine and serotonin receptors are cation-selective while glycine receptors are anion-selective). α1β heteromeric receptors are likely to be the predominant synaptic form of glycine receptors in adult mammals. (Burzomato et al., 2004). Glycine binding increases Cl^- conductance producing superpolarization and inhibition of neuronal firing. Gephyrin (Q9NQX3) anchors glycine receptors to subsynaptic microtubules.

Several homologous bacterial LIC family members have been identified (eg, 1.A.9.8-9) Hilf and Dutzler (2008) have presented X-ray structures of a pentameric ligand-gated ion channel from the bacterium Erwinia chrysanthemi at 3.3 A resolution. This high resolution structure provides a model system for the investigation of the general mechanisms of ion permeation and gating within the LIC family.