Review
Binding, activation and modulation of Cys-loop receptors

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It is over forty years since the major neurotransmitters and their protein receptors were identified, and over twenty years since determination of the first amino-acid sequences of the Cys-loop receptors that recognize acetylcholine, serotonin, GABA and glycine. The last decade has seen the first structures of these proteins (and related bacterial and molluscan homologues) determined to atomic resolution. Hopefully over the next decade, more detailed molecular structures of entire Cys-loop receptors in drug-bound and drug-free conformations will become available. These, together with functional studies, will provide a clear picture of how these receptors participate in neurotransmission and how structural variations between receptor subtypes impart their unique characteristics. This insight should facilitate the design of novel and improved therapeutics to treat neurological disorders. This review considers our current understanding about the processes of agonist binding, receptor activation and channel opening, as well as allosteric modulation of the Cys-loop receptor family.

Section snippets

Cys-loop receptor superfamily

The main pentameric receptors within the Cys-loop superfamily are nicotinic acetylcholine (nACh), 5-hydroxytryptamine (type 3, 5HT3), γ-aminobutyric acid (type A and C, GABAA/C) and glycine receptors 1, 2, 3. nACh receptors and 5HT3 receptors contain cation-selective channels that depolarize (excite) neurons, whereas GABAA/C receptors and glycine receptors contain anion-selective channels that (in most cases) hyperpolarize (inhibit) neurons, except during early postnatal development when the Cl

Cys-loop receptors: the binding step

The first step towards activation is binding of the neurotransmitter (agonist). This causes the binding site to adopt a conformation(s) that stabilizes the channel in the open state (Box 1). Binding takes place at the interface between adjacent ECDs, between loops A–C on the principal (P) face of one subunit and loops D–F on the complementary (C) face of the next [12] (Figure 1, Figure 2). Not all subunits contribute to the binding process, so the number of binding sites varies between Cys-loop

Reorganization of the binding site: loop-C capping and receptor priming

Agonist-induced reorganization of the binding site is required to transmit the binding step downstream to trigger channel opening. Such a change has been resolved in AChBPs involving a rigid body ‘capping’ motion of loop-C that closes the binding site cavity, thereby trapping bound agonist molecules [25]. Residues at the apex of loop-C move inwards by as much as 7 Å (Figure 2) 19, 25. Loop-C adopts an uncapped conformation in the absence of agonist or in the presence of antagonists, such as

Concerted reorganization of the agonist binding site during activation

Although AChBP has proved useful in identifying an agonist-induced motion of loop-C, other loop motions remain undetected. This may be because the topology of AChBP, whether unbound, agonist-bound or antagonist-bound, fits most closely to nACh receptor subunits in an ‘active-like’ state [45] and, as such, loop motions that might trigger this ‘active-like’ conformation will already be factored into the structure. Based on the 4-Å structure of the nACh receptor, loop-B, which underpins cation–π

Subunit–subunit interfaces: breaking and realigning during activation

Comparison of low-resolution nACh receptor structures with or without agonist reveal rotation of agonist-bound subunits [11]. This rotation presumably requires realignment of the binding loops between adjacent subunits. Muscle nACh receptors have charged residue pairs that bridge opposing faces of the binding site between loop-B and β5/β6-strands adjoining loop-E, which are predicted to stabilize the closed receptor and be severed upon activation [11]. Other bridging interactions within the

Rigid body motions in Cys-loop receptors

Structurally, receptors can be considered to be composed of rigid blocks [66]. Subtle movements of flexible linkers connecting these blocks allows for efficient transmission of conformational changes over distance. For instance, bending at the hinge of loop-C in AChBP can swing the rigid loop to move residues at the tip by as much as 7 Å [25].

Electron micrographs of nACh receptors identify the inner and outer β-sheets of the ECDs as potential rigid domains [11]. At high resolution without

Coupling between extracellular and transmembrane domains

For the binding signal to reach the channel it must pass from the base loops of the ECDs across to the α-helices of the TMDs (Figure 3). At the base of the ECD, the β1-2 loop of the inner β-sheet and the Cys-loop connecting the inner and outer β-sheets (connecting the bottom of the β6-strand to the β7-strand) are ideally located to communicate with the channel [11]. For nACh receptor α-subunits, these two loops form arcs using their side-chains over the M2-3 loop of the TMD, with the β1-2 loop

Cys-loop receptors: the ion channel

The final crucial step in the activation process involves channel opening. The channel is protected by a ‘gate’ that, once opened, permits a selective flux of ions across the membrane via passage through one or more selectivity filters. Electron microscopic images of the nACh receptor pore at 4-Å resolution [91] reveal that the channel comprises a ring of five vertically aligned M2 α-helices, each one contributed by a separate subunit. The helices are not perfectly parallel; the extracellular

Location of the channel gate

In the closed nACh receptor conformation, a constriction in the channel lumen at 9′–14′ is evident from electron micrographs [11]. It is at this midpoint, around the M2 kink, that the α-helices draw together to form the principal gate. Two rings in particular, 9′ leucines and 13′ valines, face into the lumen and constrict the channel by forming a hydrophobic girdle approximately 6 Å in diameter 11, 91, 93, 94, 95. This constriction is sufficient to block the passage of hydrated Na+ and K+ ions,

Opening the channel gate

Based on low-resolution images of nACh receptors in a presumed open state, channel opening was originally thought to occur by sideways rotation of the M2 α-helices, swinging the kinks of the hydrophobic girdle outwards [11]. However, a helical tilting motion is favoured by two functional studies; one measuring changes in channel conductance caused by protonation of basic residues introduced into the channel [106], and the other measuring the interaction of Zn2+ in the channel with substituted

Ion selectivity filter

The charged rings that exist at the cytoplasmic and extracellular ends of the channel are vital for regulating channel conductance (Figure 5). These rings are negatively charged in nACh receptors and 5HT3 receptors and selective for cations, whereas they are positively charged for the anion-permeable GABAA/C receptors and glycine receptors [108]. Sequentially reversing the charges in these rings for nACh receptors, GABAA receptors and glycine receptors has revealed an inverse relationship

Modulation of the transmembrane domain

The transmembrane domains of Cys-loop receptors, particularly GABAA receptors, contain binding sites for a range of clinical and endogenous modulators [130]. Numerous residues are known to affect the activities of drugs and modulators, but the 15′ residue of M2 is often a key determinant. This residue faces away from the ion channel and into a water-filled cavity that appears capable of accommodating drugs 75, 91, 109 (Figure 6). As such, the 15′ residue is ideally located to bind drugs

Conclusions and future directions

Significant progress has been made towards our understanding of the operating principles behind the activation and modulation of the Cys-loop receptor. The overall view of loop-C capping, the formation of pre-activated states and the retraction of the M2 helices to open a hydrophobic gate are becoming quite established, whereas other concepts such as the concerted reorganisation of agonist binding loops and the molecular mechanisms underlying transmembrane modulators have yet to be

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