General

The glycine receptor Cl^- channel (GlyR) is known for mediating inhibitory synaptic transmission between interneurons and motor neurons in reflex circuits of the spinal cord. Glycine was originally proposed as an inhibitory neurotransmitter based on an analysis of its distribution in the spinal cord [1]. Subsequent experiments showed that it activated a strychnine-sensitive inhibitory Cl^- conductance in spinal cord neurons [3-4,18]. Purification of the GlyR from rat spinal cord by strychnine affinity chromatography revealed three distinct polypeptides of molecular mass 48, 58 and 98 kDa [14]. The 48 and 58 kDa peptides were later shown to correspond to the α1 and β subunits, respectively. Cloning of the α1 GlyR subunit was reported in 1987 [7] and its homology with the nicotinic acetylcholine receptor led to its inclusion in the Cys-loop family of ligand-gated ion channel receptors, now known as the pentameric ligand-gated ion channel family. The α2, α3 and α4 subunits, as well as several splice variants, were subsequently cloned by homology screening [12]. GlyRs mediate inhibitory neurotransmission in the spinal cord, brainstem and retina. They are also found pre-synaptically, where they modulate neurotransmitter release. Non-synaptic GlyRs exist in neurons in most parts of the brain, as well as in sperm and macrophages. GlyRs conduct monovalent halide anions in a non-selective manner and exhibit unitary conductances of ~40-90 pS.

Structure

GlyRs are pentameric assemblies where the five subunits are arranged symmetrically around a central ion-conducting pore. High resolution molecular structures are available for the α1 and α3 homomeric GlyRs [5,8] and for the αβ heteromeric GlyR [10]. These and other structural studies have provided detailed information concerning the pharmacology of ligand binding and the mechanisms of GlyR channel gating. Like other Cys-loop receptor subunits, each GlyR subunit comprises a large extracellular amino-terminal domain that includes the ligand binding sites which connects to a bundle of four α-helical transmembrane segments (termed M1-M4) with a large intracellular domain between M3 and M4. Each of the five subunits contributes an amphipathic M2 domain to the lining of the central water-filled pore. All four α subunits (α1-α4) express robustly as homomeric GlyRs in vitro, although β subunits express only as αβ heteromers with a putative 4α:1β stoichiometric ratio [20]. The β subunit serves the important role of anchoring GlyRs to postsynaptic densities via the cytoplasmic clustering protein, gephyrin [6]. In the rat, homomeric α1, α3 and α4 GlyRs are weakly expressed at all developmental stages, although homomeric α2 GlyRs are abundantly expressed in embryonic neurons only. The majority of glycinergic neurotransmission in adults is mediated by heteromeric α1β GlyRs.

Pharmacology

Many small amino acids activate GlyRs, and they are all less potent and efficacious than glycine itself. Agonists are not useful in GlyR classification as the rank orders of agonist potency and efficacy are similar across different GlyRs. Among several agonists, aminomethanesulfonate (unstable at neutral pH), L-alanine, L-serine and β-alanine are the most efficacious, whereas taurine is only a partial agonist and GABA is a very weak partial agonist [9]. Most commercially-available agonists are contaminated by glycine and should be purified before use.

The only non-peptidic agonist identified so far is ivermectin [16]. GlyRs are potently and selectively antagonised by the classical competitive antagonist, strychnine. Although many compounds exhibit modest subunit-specific pharmacological differences at GlyRs [2,17] to date there are few substances with sufficient discriminatory capacity to identify the presence of α1, α2, and α3 subunits in either homomeric or heteromeric GlyRs. Although picrotoxin distinguishes strongly between α homomeric and αβ heteromeric GlyRs [15] and synthetic cannabinoid agonists (WIN55212-2, HU210 and HU308) distinguish α1 from α2 or α3 containing GlyRs [19], unfortunately all these agents also have potent effects on other receptor types, which limits their utility as probes for establishing the physiological role of different GlyR subtypes. Notably, Zn²⁺ acts as a concentration-dependent (nanomolar) potentiator and inhibitor (micromolar) at GlyRs [11,13]. There is thus abundant scope for the development of novel GlyR-specific agents as both therapeutic leads for movement disorders and chronic inflammatory pain and as subunit-specific pharmacological probes for basic research.

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