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School of Biomedical Sciences, University of Queensland, Brisbane, Australia
ABSTRACT I. INTRODUCTION A. Scope of This Review B. Glycine as an Inhibitory (and Excitatory) Neurotransmitter II. DIVERSITY, DISTRIBUTION, AND FUNCTION OF GLYCINE RECEPTOR SUBUNITS A. Molecular Diversity B. Distribution and Function in the Rat Nervous System 1. Distribution of functional GlyRs A) DISTRIBUTION OF STRYCHNINE AND GLYCINE BINDING SITES. B) DISTRIBUTION OF GLYR IMMUNOREACTIVITY. C) DISTRIBUTION OF FUNCTIONAL GLYCINERGIC SYNAPSES. 2. Distribution of GlyR subunits 3. Developmental switch from {alpha}2 to {alpha}1{beta} 4. A special case: the retina C. Distribution and Function in Other Tissues 1. Spermatozoa 2. Endocrine pancreas 3. Adrenomedullary chromaffin cells 4. Kupffer cells and other macrophages 5. Neural stem progenitor cells III. STRUCTURE AND ASSEMBLY A. General Structural Features B. Transmembrane Domains 1. Spatial organization 2. Methods for probing TM domain secondary structure A) SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD. B) TRYPTOPHAN SCANNING MUTAGENESIS. C) HYDROPHOBIC REAGENT REACTIVITY. 3. TM1 4. TM2 5. TM3 6. TM4 C. NH2-Terminal Ligand-Binding Domain 1. Structural homology with AChBP 2. Glycosylation D. Large Intracellular Domain 1. Ubiquitination domain 2. SH3-binding motif 3. Phosphorylation sites 4. Gephyrin binding domain 5. Basic cluster required for TM3 integration E. Receptor Assembly 1. Subunit stoichiometry and arrangement 2. Intersubunit contact points and assembly domains 3. Effects of high receptor density 4. Coassembly with other LGIC subunits IV. STRUCTURE AND FUNCTION OF THE PORE A. Functional Properties of the Pore 1. Ionic selectivity 2. Single-channel conductance B. Molecular Determinants of Ion Selectivity and Conductance C. The Channel Activation Gate D. Molecular Mechanisms of Desensitization V. AGONIST BINDING AND RECEPTOR ACTIVATION A. Introduction 1. Review of basic receptor theory 2. Concerted versus sequential modelsof receptor activation B. Kinetic Models of Glycine Receptor Gating C. Agonist Binding 1. Agonist affinity and efficacy 2. Agonist binding domains 3. Physical basis of agonist binding D. Structural Changes Accompanying Activation 1. The ligand-binding domain 2. The TM2-TM3 domain 3. The TM1-TM2 domain 4. The membrane-spanning domains 5. The {beta}-subunit VI. GLYCINE RECEPTOR MODULATION A. Phosphorylation 1. Protein kinase A 2. Protein kinase C 3. Protein tyrosine kinase B. Modulators of Possible Physiological Relevance 1. Zinc A) A PUTATIVE PHYSIOLOGICAL ROLE. B) MOLECULAR MECHANISM OF ACTION. C) INHIBITION. D) POTENTIATION. 2. Calcium 3. pH 4. Neurosteroids 5. G protein {beta}{gamma}-subunits C. Molecular Pharmacology 1. Strychnine and analogs 2. Picrotoxin and analogs 3. Cyanotriphenylborate 4. Ginkgolides 5. Tropisetron and other 5-HT3R antagonists 6. Ivermectin 7. Alcohols, anesthetics, and inhaled drugs of abuse 8. Miscellaneous bioactive compounds VII. GLYCINE RECEPTOR CHANNELOPATHIES A. Human Startle Disease B. Murine Startle Syndromes C. Bovine Myoclonus VIII. OUTLOOK ACKNOWLEDGMENTS REFERENCES
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-subunits (1 to 4) and one 
-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from 2 to 1 is completed by around postnatal day 20 in the rat. The -subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning GlyR molecular structure and function. Most of the information has been obtained from homomeric 1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs. |
I. INTRODUCTION |
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Ligand-gated ion channels permit cells to respond rapidly to changes in their external environment. They are particularly well known for mediating fast neurotransmission in the nervous system. The glycine receptor (GlyR) is a membrane-embedded protein that contains an integral Cl–-selective pore. When glycine binds to its site on the external receptor surface, the pore opens allowing Cl– to passively diffuse across the membrane. The GlyR is a member of the pentameric ligand-gated ion channel (LGIC) family, of which the nicotinic acetylcholine receptor cation channel (nAChR) is the prototypical member. Other members of this family include the cation-permeable serotonin type 3 receptor (5-HT3R), anion-permeable GABA type A and C receptors (GABAAR and GABACR), recently identified cation-permeable zinc and GABA receptors (34, 86), as well as invertebrate anion-permeable glutamate and histidine receptors (130). Note that glycine also directly activates a cation-selective ion channel of the excitatory glutamate receptor family (62). The structural and functional properties of this receptor class have recently been reviewed (94) and are not considered here.
Glycine was first proposed as an inhibitory neurotransmitter on the basis of a detailed analysis of its distribution in the spinal cord (16). Subsequent electrophysiological studies demonstrated a strychnine-sensitive hyperpolarizing action of glycine on spinal neurons (80, 395). This hyperpolarization was soon discovered to be mediated by an increase in Cl– conductance (81, 82, 396). The receptors responsible for these actions were subsequently purified by strychnine affinity chromatography (291, 292), and the first GlyR subunit was cloned in 1987 (138).
Current research into the GlyR can be divided into two major strands. The first involves the investigation of the molecular mechanisms of GlyR trafficking and clustering at synapses. This area is currently the subject of intense investigation, and recent progress has been covered in several authoritative reviews (189, 190, 219). The second research strand is concerned with understanding the molecular structure and function of the GlyR. Research has intensified in this area over the past few years, and the purpose of this review is to present a coherent view of recent findings. Much of our understanding of GlyR structure-function has been gained by comparison with the structurally homologous nAChR. Hence, this review makes frequent references to research on the nAChR, particularly in areas where knowledge of the GlyR is deficient.
B. Glycine as an Inhibitory (and Excitatory) Neurotransmitter
When the GlyR is activated, the resulting Cl– flux moves the membrane potential rapidly toward the Cl– equilibrium potential. Depending on the value of the equilibrium potential relative to the cell resting potential, the Cl– flux may cause either a depolarization or a hyperpolarization. The GlyR is generally known as an inhibitory receptor because the Cl– equilibrium potential is usually close to or more negative than the cell resting potential. Subthreshold depolarizations can inhibit neuronal firing if they are accompanied by an increase in membrane conductance that shorts out excitatory responses, a phenomenon termed "shunting inhibition." However, in embryonic neurons, the intracellular Cl– concentration is raised substantially, with the effect that GlyR activation causes a strong, suprathreshold depolarization. These large glycine-induced depolarizations gate a calcium influx that is necessary for the development of numerous specializations, including glycinergic synapses (190). The switch to the mature neuron phenotype is mediated by the expression of a K+-Cl– cotransporter, KCC2, which lowers the internal Cl– concentration, thereby shifting the Cl– equilibrium potential to more negative values and converting the actions of the GlyR from excitatory to inhibitory (355).
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II. DIVERSITY, DISTRIBUTION, AND FUNCTION OF GLYCINE RECEPTOR SUBUNITS |
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Betz and colleagues (291, 292) originally purified the rat spinal cord GlyR by affinity chromatography on aminostrychnine-agarose columns. Oligonucleotides designed from peptide sequences of purified receptors were then used to probe a rat spinal cord cDNA library, resulting in isolation of cDNA clones corresponding to the 48 kDa (1) and 58 kDa () subunits (137, 138). Subsequently, cDNAs of the rat 2- and 3-subunits were cloned by homology screening (9, 199, 201). The 4-subunit, which does not appear to exist in the rat or human, was first identified in the mouse (254) and has subsequently been found in the chick (157) and zebrafish (91). While the -subunit genes are highly homologous, with primary structures displaying 80–90% amino acid sequence identity, the -subunit has a sequence similarity of 
47% with the 1-subunit (137).
The rat 1-subunit has a splice variant, termed 1ins, which contains an eight-amino acid insert in the large intracellular loop (244) that contains a possible phosphorylation site (see sect. VIA). Alternative splicing of the rat 2-subunit generates two splice variants, 2A and 2B (199, 201). The 2B-variant differs from 2A by the V58I and T59A amino acid substitutions. Another version of the 2-subunit, termed 2*, incorporates a single amino acid substitution (G167E) that confers strychnine insensitivity (202). Two transcripts of the human 3-gene, termed 3L and 3K, have been characterized (280). The 3K-variant lacks a 15-amino acid segment in the large intracellular loop that exists in both the rat 3- and the human 3L-subunits. A splice variant of the zebrafish 4-subunit contains a 15-amino acid insert in the ligand-binding domain (91). Although a human -subunit gene polymorphism has been described (264), it does not appear to result in a coding mutation.
All GlyR genes cloned to date share a similar exon-intron organization with the coding region spread over nine exons (254, 264, 346). This common organization suggests a phylogenetic gene duplication (345).
B. Distribution and Function in the Rat Nervous System
1. Distribution of functional GlyRs
A) DISTRIBUTION OF STRYCHNINE AND GLYCINE BINDING SITES. Autoradiographic localization of [3H]strychnine binding sites was first studied by Zarbin et al. (431) in the rat. Strychnine binding sites were shown to exist at high levels in the spinal cord and medulla and at lower levels in the pons, thalamus, and hypothalamus, while being virtually absent in higher brain regions. In the spinal cord, their distribution is relatively diffuse throughout the gray matter. In contrast, GlyRs in the brain stem are highly localized to discrete nuclei, notably the trigeminal nuclei, the cuneate nucleus, the gracile nucleus, the hypoglossal motor nucleus, the reticular nuclei, and cochlear nuclei (301). The retina, which also has a high concentration of strychnine sites (297), is considered as a separate case, below. Together, these areas comprise a subset of the distribution as determined by glycine autoradiography (270) or glycine immunoreactivity (310), a mismatch that is understandable given that glycine is also associated with glutamatergic synapses (270). An advantage of strychnine autoradiography is that it reveals the presence of surface-expressed receptors, but a disadvantage is that its limited resolution does not permit the ultrastructural localization of strychnine binding sites. Thus strychnine autoradiography does not necessarily define the distribution of glycinergic synapses.
B) DISTRIBUTION OF GLYR IMMUNOREACTIVITY.
Many studies have examined the immunocytochemical localization of GlyRs at the light and electron microscopic levels using generic GlyR -subunit monoclonal antibodies. At the light microscopic level, there is a strong correlation between the distribution patterns revealed by GlyR immunoreactivity and strychnine autoradiography (17, 370). However, some significant differences have been observed. First, immunolabeling reveals the existence of GlyRs in the cerebellum (17, 361) and olfactory bulb (383), whereas none was seen using strychnine binding (431). Second, the substantia gelatinosa in the spinal cord is strongly labeled by strychnine (431), but not by GlyR antibodies (23). The reasons for the differential labeling are yet to be clarified.
Electron microscopic immunoreactivity reveals that GlyRs at central synapses are concentrated into regions closely apposed to presynaptic terminals (13, 23, 370, 371, 383), strongly suggesting a functional role. It is of interest to note that this approach has demonstrated the colocalization of GlyRs and GABAARs at individual postsynaptic densities in the spinal cord (43, 127, 369) and cerebellum (100).
C) DISTRIBUTION OF FUNCTIONAL GLYCINERGIC SYNAPSES.
Most central nervous system neurons are inhibited by glycine (279). Of course, the mere presence of functional GlyRs, especially on dissociated or cultured neurons, does not imply a physiological role. However, it has recently been proposed that the activation of nonsynaptic GlyRs in embryonic cortical neurons may be important for development, and that taurine released from local glial cells may be the endogenous ligand (114). Similarly, nonsynaptic GlyRs on hippocampal CA3 neurons are proposed to be held in a tonically active state by locally released taurine or -alanine (273). The idea that glycinergic ligands may act on nonsynaptic GlyRs to mediate processes of physiological importance certainly warrants further attention. Traditionally, however, a functional role for GlyRs in neurons has required the demonstration of strychnine-sensitive synaptic currents.
There is abundant evidence for the existence of functional glycinergic synapses in the retina (see below) in spinal cord motor reflex pathways (219) and in spinal cord pain sensory pathways (4). Glycinergic neurotransmission has also been demonstrated in various brain stem nuclei. For example, it has been well characterized in several brain stem nuclei of the central auditory pathways. In the medullary cochlear nucleus, which receive inputs directly from the auditory nerve, glycinergic synapses occur onto stellate cells (108) and bushy cells (230). In the trapezoid body, a subsequent major relay station in the auditory pathway, GlyRs are located presynaptically at calyceal synapses onto principal cells (373). The prominent output from this nuclei extends to the superior olivary complex of the pons, where glycinergic synapses are also found (194, 351). Functional glycinergic synapses also exist on neurons in the medullary trigeminal (421), abducens (325), and hypoglossal motor nuclei (248, 347, 375). In the cerebellum, glycinergic synapses mediate inhibitory neurotransmission between Lugaro cells and Golgi cells in the cerebellar cortex (93), and between interneurons and principal cells in the deep cerebellar nuclei (182). This list may expand as other brain stem nuclei are characterized in detail.
It is relevant to note that glycine may not be the sole inhibitory neurotransmitter at many of these synapses. Mixed GABA-glycine synapses may mediate neurotransmission at individual synapses in the spinal cord (174), brain stem (194, 283, 325), and cerebellum (100). Interestingly, GlyR activation appears to be able to inhibit GABAARs via a phosphorylation-dependent mechanism (229). This process may be important for regulating inhibitory synaptic current magnitude at mixed GABA-glycine synapses. There is evidence that the GABAergic component of inhibitory neurotransmission at mixed synapses may be upregulated in individuals suffering from heritable disorders of glycinergic neurotransmission (see sect. VII).
Finally, presynaptic GlyRs have been functionally characterized at calyceal synapses (373) and on terminals synapsing onto rat spinal sensory neurons (172). Surprisingly, in both cases GlyR activation is excitatory, leading to increased neurotransmitter release.
2. Distribution of GlyR subunits
In situ hybridization was the first approach employed to localize the distribution of individual GlyR subunits in the rat. An advantage of this approach is its subtype specificity, but a disadvantage is that transcript expression does not necessarily imply the surface expression of functional receptors. Expression of 1-subunit mRNA in adult rats was highest in the brain stem nuclei and spinal cord, but it was also found in the superior and inferior colliculi and in regions of the thalamus and hypothalamus (245, 331). It was notably absent from cortical regions. Thus, with few exceptions, its distribution is similar to that of functional GlyRs as described above. In the rat, expression is detectable at embryonic day 15 and increases to a maximum at around postnatal day 15, without substantial changes in its distribution (245). Northern blot analysis reveals that 1ins shares a similar distribution (244).
Prenatally, transcripts of the 2-subunit gene are found throughout most of the central nervous system. However, postnatally they decline sharply with little label remaining by postnatal day 20 (9, 245, 392). Detectable amounts of 2-transcripts do persist into adulthood, however, notably in the retina (see below), auditory brain stem nuclei (293), and some higher brain regions (245). The 2A-isoform is expressed more abundantly than 2B during development, although the 2B-isoform is present at higher levels in the adult (199).
The distribution and developmental changes in 3-transcripts generally resemble those of 1-transcripts, with the exception that 3-expression is much less intense at all developmental stages (245). As with the 1-subunit, its expression intensity increases postnatally to reach a maximum at around 3 wk (245). The 3L- and 3K-variants share similar distribution patterns (280). GlyR 4-subunit transcripts are expressed at very low levels (if at all) in the adult rat (293), although they are strongly expressed in the spinal cord, dorsal root ganglia, sympathetic ganglia, and the male genital ridge of the chick (157).
GlyR -subunit transcripts are distributed widely throughout the embryonic and adult central nervous system (125, 245). Although present at low levels prenatally, expression increases dramatically after birth and persists into adulthood (245). The reason for this broad expression profile is somewhat puzzling given that these subunits do not form functional receptors in the absence of -subunits.
3. Developmental switch from 2 to 1
Becker et al. (27) showed using protein expression that fetal GlyRs are predominantly 2-homomers, whereas adult receptors are predominantly 1-heteromers. Such a switch is also supported by the mRNA expression patterns described above. In the neonatal rat, the 1-, 2-, and -subunits exist in abundance, implying a mixture of receptor isoforms, but the switch towards the adult isoform is complete by around postnatal day 20 (27, 121, 392). The sparse expression of the 3- and 4-subunits suggests they may also be included in a minority of adult GlyRs. Recent evidence suggests this switch may not be as complete as originally thought and that 2-subunit expression may remain at significant levels throughout adulthood in the retina (see below) and auditory brain stem (293). Although the mechanism responsible for triggering the developmental switch is not known, it does not seem to require the activation of the GlyRs themselves (247).
Given that 2-subunits alone are expressed in embryonic neurons, is it possible that homomeric 2-GlyRs may mediate synaptic transmission? Takahashi et al. (361) showed that the single-channel conductance and kinetic properties of recombinant homomeric 2- and 1-GlyRs were similar to those of native GlyRs in rat spinal neurons at embryonic day 20 and postnatal day 22, respectively. They also demonstrated an increased decay rate of the glycinergic inhibitory postsynaptic currents (IPSCs) over the same period that was consistent with the change in channel kinetic properties (361). Subsequent studies have supported these findings (12, 347). However, it is unlikely that homomeric 2-GlyRs mediate inhibitory neurotransmission for the following reasons. First, because -subunits are required for GlyR postsynaptic clustering (188, 259), it is not certain how the 2-homomers would undergo the prerequisite aggregation at postsynaptic densities. Second, a recent study has found that 2-homomeric GlyRs activate too slowly to effectively mediate synaptic transmission (246). Given their wide distribution throughout the nervous system during development, and the fact that Cl– fluxes are excitatory in developing neurons (114, 355), it seems more likely that homomeric 2-GlyRs mediate nonsynaptic cell-to-cell communication that could be important for neuronal differentiation and synaptogenesis (190). Glycinergic synapses in immature neurons are probably comprised of 2-heteromeric GlyRs (219). The single-channel conductance of synaptic GlyRs from embryonic neurons is consistent with such a conclusion (12, 347).
This is considered separately because the profile of GlyR subunit distribution is atypical and has been mapped in detail and because a specific role for glycinergic synapses has been proposed. GABA and glycine both function as inhibitory neurotransmitters in the retina (143, 176, 297, 390). In situ hybridization and immunohistochemistry both show that GlyR 1-, 2-, 3-, and -subunits have different patterns of distribution in the adult rat (136, 146, 330). The 1-subunit is distributed predominantly on bipolar cells and on some ganglion cells in the inner plexiform layer (136, 144, 145, 231). The 2-subunit is distributed on amacrine cells and on almost all ganglion cells, whereas the 3- and -subunits are distributed widely throughout the inner plexiform layer (136). A detailed study in the mouse concluded that 1-subunits are associated with synapses in the rod pathway between AII amacrine cells and off-cone bipolar cells, whereas 3-subunits are restricted to cone pathways (159). Together these results indicate a spatial distribution of GlyR subunit composition throughout the adult retina. Consistent with this picture, Enz and Bormann (105) detected mRNA for all four GlyR subunits in RNA from whole retina, but mRNA for only 1- and -subunits in RNA isolated from individual rod bipolar cells.
Electron microscopy has confirmed that the punctate immunoreactivity seen with the light microscope is due to clusters of GlyRs at the postsynaptic densities (146, 330). Consistent with these anatomical studies, electrophysiological investigations in the rat have revealed the presence of glycinergic inhibitory postsynaptic potentials (IPSPs) in identified amacrine cells (119), ganglion cells (153, 302, 368), and rod bipolar cells (77, 99, 303).
The synaptic distribution of GlyR subunits is spatially distinct from that of GABAAR subunits, although individual ganglion cells may possess both types of synapse (195, 329). Recent studies have begun to address the possibility that glycinergic and GABAergic transmission may have distinct physiological roles. Although functional differences between GABAergic and glycinergic IPSPs in retinal neurons have been demonstrated (119, 302), the physiological significance remains unknown. However, structural studies have provided evidence that the GABA and glycine synaptic pathways participate in different functional circuits. In particular, glycinergic synapses are thought to play a specific role in the transmission of dark-adjustment signals through the off-channel of the rod pathway from amacrine cells to off-bipolar cells and hence to off-ganglion cells (146, 330, 391). This circuit contributes to the switch from day to night vision.
C. Distribution and Function in Other Tissues
The front of the mammalian sperm head contains a large secretory vesicle termed the acrosome. The process of fertilization is initiated when the sperm head contacts the outer coat, or zona pellucida, of the egg. A zona pellucida-specific glycoprotein, ZP3, forms the sperm receptor. Its interaction with sperm initiates a complex intracellular signaling mechanism inside the sperm that culminates in a calcium elevation that is thought to be mediated at least partly by an influx through voltage-gated calcium channels (115). This event, termed the acrosome reaction (AR), results in the release of acrosome hydrolytic enzymes by exocytosis. These enzymes induce various protein modifications to ensure that the sperm remains tightly bound to the zona pellucida while fusion takes place between the sperm and egg plasma membranes.
The activation of GlyRs and GABAARs in the sperm plasma membrane appears to be essential for the AR (257). There is considerable evidence that GlyRs exist in sperm plasma membranes. For example, immunochemical studies have demonstrated the existence of GlyR - and -subunit protein in porcine, mouse, and human sperm (49, 258, 332). An immunofluorescence study localized the -subunits to cell membranes in the periacrosomal regions of live mouse sperm (332). In addition, strychnine binding studies have revealed the presence of GlyRs in hamster sperm (232).
Functional evidence for GlyR involvement has also been demonstrated. For example, glycine initiated the AR in a manner that was inhibited by strychnine or a GlyR -subunit antibody (49, 332). Furthermore, studies using fura 2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the ZP3-mediated calcium influx (49). Finally, sperm from homozygous spasmodic and spastic mice (which possess defective GlyR 1- and -subunits, respectively) are deficient in their ability to undergo the AR (333).
Thus the GlyR is likely to have a central role in the AR. However, two questions remain about this process. What is the concentration of Cl– inside sperm? Presumably, it must be high enough to force an outward (i.e., depolarizing) Cl– flux upon GlyR activation. Second, what is the glycine concentration in the oviduct where fertilization takes place? Do the GlyRs remain tonically active holding the sperm in a depolarized state preceding the AR?
Harvey et al. (157) found that the 4-subunit gene is expressed on the developing male genital ridge of the chick and proposed that GlyRs containing this subunit may contribute to the development of immature spermatogonia.
A pancreatic cell line, GK-P3, expresses functional GlyRs. When activated, these receptors cause a depolarization that increases the intracellular calcium concentration (393). A glycine receptor antibody displayed immunoreactivity with GK-P3 cells and with isolated rat pancreatic islet cells (393) prompting the authors to surmise that GlyRs may also be expressed in islet cells in vivo. However, there is as yet no electrophysiological evidence for GlyRs in pancreatic islet cells.
3. Adrenomedullary chromaffin cells
High-affinity [3H]strychnine binding sites have been shown to exist in catecholamine-secreting chromaffin cells of the adrenal medulla (415, 416). The same group subsequently demonstrated that glycine can stimulate significant catecholamine secretion from chromaffin cells in both in vitro and in vivo assays (414, 417). The presence of GlyR 3-subunit mRNA (but not 1 or 2) was also demonstrated by RT-PCR from RNA extracted from rat adrenal glands. However, direct electrophysiological evidence for glycine-activated currents in chromaffin cells is conspicuously absent to date.
4. Kupffer cells and other macrophages
A variety of pharmacological evidence, summarized in Reference 184, suggests that GlyRs may at least partially mediate the anti-inflammatory effects of glycine in macrophages and leukocytes. Research on GlyR involvement in these processes has focused on Kupffer cells, which are specialized macrophages found in the liver. Glycine has been shown to reduce the magnitude of lipopolysaccharide-induced calcium transients in these cells in a strychnine-dependent manner (122, 167). Similar observations have also been made in neutrophils (398) and hepatic parenchymal cells (304). Recent evidence from Western blots, RT-PCR, and RNAse protection assays indeed suggest the presence of GlyR 1-, 2-, 4-, and -subunits in Kupffer cells (122).
5. Neural stem progenitor cells
Strychnine-sensitive glycine-gated currents are present in postnatal, nestin-positive neural stem progenitor cells (278). Consistent with this observation, RT-PCR and immunocytochemical methods revealed the presence of 1-, 2-, and -subunit RNA transcripts and -subunit protein, respectively.
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The nAChR is the most intensively investigated member of the LGIC family. Consequently, most of the structural features of the GlyR have been deduced from its homology with this receptor. LGIC receptors contain five subunits arranged pseudo-symmetrically around a central ion-conducting pore. The membrane topologies of all LGIC subunits are similar. This topology includes a large NH2-terminal extracellular domain that contains the agonist binding sites. A defining feature of LGIC subunits is the conserved cysteine loop in this domain. All GlyR subunits also harbor a second cysteine loop (309) that incorporates a principal glycine-binding domain. As discussed in detail below, the crystal structure of acetylcholine-binding protein (AChBP) provides an excellent model for understanding the structure of this domain (52).
Hydropathy analysis originally predicted an arrangement of four -helical transmembrane domains (TM1–TM4) per subunit. Although evidence exists for the inclusion of -sheet in the TM regions (134), the recent elucidation of the crystal structure of the Torpedo nAChR TM domains provides an overwhelming argument in favor of the original four -helical model (267). This structure, determined by cryoelectron microscopy to a resolution of 4 Å by Miyazawa et al. (267), is a major advance in the field. Finally, TM3 and TM4 are linked by a large, poorly conserved, intracellular domain that contains phosphorylation sites and other sites for mediating interactions with cytoplasmic factors. The structure and function of each of these regions is now considered in detail.
The principal role of the TM domains is to provide a sealed barrier to separate the ion permeation pathway from the apolar region of the lipid bilayer. In most ion channels of known structure, this is achieved by a close packing of amphipathic -helices at angles close, but not quite perpendicular, to the plane of the membrane (353). This arrangement also applies to LGIC receptors, with each subunit contributing an -helical TM2 domain to the lining of a single central water-filled pore. The TM1, TM3, and TM4 domains surround TM2 and provide the interface with the lipid bilayer, thereby isolating TM2 from direct contact with the surrounding hydrophobic environment. Viewed from the synapse, TM1–TM4 are arranged consecutively in a clockwise manner, with TM1 and TM3 located closest to TM2 (267). In the nAChR, the TM domains splay outwards towards the extracellular membrane surface and extend about two helical rotations (10 Å) beyond the hydrophobic membrane core. As noted by Miyazawa et al. (267), the extracellular spaces between the splayed helices appear to afford a lateral pathway (in addition to the large central outer vestibule pathway) for ions to access the pore.
The remainder of this section attempts to relate the Miyazawa TM domain structure with an abundance of earlier information that also bears upon TM domain structure and function. However, before doing so, it is worth briefly considering three functionally based techniques that have been of particular value in defining the secondary structure of ion channel pore-lining domains.
2. Methods for probing TM domain secondary structure
A) SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD.
The substituted cysteine accessibility method (SCAM) was initially applied as a means of identifying the secondary structure of ion channel pore-lining domains (5, 8). The method entails introducing cysteine residues one at a time into the protein domain of interest. Cysteine reactivity is then assayed by exposure to highly soluble, sulfhydryl-specific reagents, generally methanethiosulfonate (MTS) derivatives (180). If a functional property of the channel is irreversibly modified upon exposure to such a reagent, the cysteine is assumed to be exposed at the water-accessible protein surface. If every second residue is reactive, then the secondary structure is interpreted as -sheet (8), whereas if every third or fourth residue is exposed, the structure is interpreted as -helical (7). This approach is now applied more widely to probe structural changes in extramembranous domains (e.g., Ref. 234). However, a drawback of applying this approach outside the pore is that a lack of functional modification does not necessarily mean that the residue has not reacted. In other words, negative results cannot be interpreted. However, this limitation is less likely to apply in the spatially restricted environment of an ion channel pore, where attachment of a large side chain is more likely to affect current flow, thus providing a generally more reliable measure of cysteine reactivity. Various extensions to this technique have also proven useful. For example, by determining changes in cysteine reactivity in various functional states (e.g., closed, open, and desensitized), it may be possible to draw conclusions about state-dependent structural changes. Similarly, by comparing state-dependent reaction rates of positively and negatively charged reagents, it may be possible to estimate the local electrostatic potential (289, 405). The originators of SCAM have provided an excellent review of its capabilities and limitations (180).
B) TRYPTOPHAN SCANNING MUTAGENESIS.
This approach involves introducing tryptophan residues one at a time into the domain of interest. Because tryptophan side chains are bulky, it is assumed that if they protrude into the relatively fluid lipid bilayer they should be less likely to disrupt receptor structure and function than if they protrude towards the protein interior (71). Experimentally, one or more basic functional properties (e.g., agonist EC50) of each mutant receptor is measured, and then a correlation is drawn between the position of the introduced tryptophan and the severity of the functional consequence. As with SCAM, any resulting periodicity is interpreted as -sheet or -helix.
C) HYDROPHOBIC REAGENT REACTIVITY. n this approach, employed extensively by Blanton and colleagues in the nAChR (22, 40, 41), labeled hydrophobic reagents are incubated with the receptor. The identity of the residues that are covalently modified by these compounds is then determined using biochemical assays. Residues thus identified are assumed to be exposed to the lipid bilayer. Again, any resulting periodicity is interpreted in terms of secondary structure.
By connecting directly with the NH2-terminal domain, TM1 is ideally located to act as a linkage between the ligand-binding site and the channel activation gate. Hence, an unequivocal understanding of its structure and relationship with TM2 is essential. The Torpedo nAChR crystal structure identifies TM1 as an -helix that is initiated at the residue corresponding to Y222 of the 1-GlyR and enters the membrane at around M227. As stated above, it is likely that water-filled space surrounds the extracellular portion of this helix. In support of this, an aqueous tyrosine-specific reagent labeled two tyrosines (Y223, Y228) in TM1 of the 1-GlyR (222). Furthermore, SCAM analysis on the nAChR revealed several extramembranous TM1 residues that are accessible to modification by hydrophilic reagents (432). Several lines of evidence implicate the extramembranous TM1 residues in LGIC gating (e.g., Refs. 6, 38, 102, 363, 432). Throughout the membrane-embedded portion of the nAChR TM1 there appears to be a distinct absence of van der Waals contacts with TM2, implying that a water-filled pocket separates the respective domains (267). However, by homology with nAChR, there may be a hydrophobic bond linking I234 (or L237) and M12' in TM2 of the 1-GlyR. At its intracellular end, the TM1 -helix is probably terminated by the aspartic acid at position 247.
Affinity labeling experiments employing pore-blocking substances first suggested an -helical open state structure of the nAChR TM2 (reviewed in Ref. 18). The Torpedo nAChR structure of Miyazawa et al. (267) confirms the long-held view that this domain forms an -helix throughout its entire length (267). As summarized in Figure 1A, an -helical structure is also strongly supported by SCAM analysis. Indeed, the luminal exposure patterns as determined by SCAM and the Miyazawa structure are entirely in agreement. SCAM also reveals a highly conserved pattern of residue exposure in the nAChR, GABAAR, and 5HT3R (Fig. 1A), suggesting that a similar pattern applies to all LGIC members.
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1-subunit residues line the pore: G2', T6', R19', and A20' (234, 341). By homology with other LGICs (7, 234, 341, 409), the following residues are also likely to line the pore: T7', L9', T10', T13', S16' and G17' (Fig. 1A). When viewed on an -helical net, these residues form a hydrophilic "strip" along one side of an otherwise hydrophobic -helix (Fig. 1B). A predicted cross-section through the 1-GlyR pore is shown in Figure 1C. The pore exposure pattern of residues intracellular to G2' cannot currently be predicted for anionic LGICs because, as discussed in detail in section IVB, they contain an additional proline at position –2' that is likely to induce structural disruptions around the internal pore boundary. Because the GlyR -subunit TM2 has an unusually low sequence homology with all other LGIC TM2 domains (Fig. 1A), it will be of interest to determine whether it also shares the consensus residue exposure pattern.
Structural analysis shows TM2 to be kinked radially inwards, attaining a minimum pore diameter at its midpoint (267). Miyazawa et al. (267) propose that this constriction facilitates a tight hydrophobic coupling between TM2 residues of neighboring subunits at two levels in the central region of the pore. The first contact is thought to occur between L9' of one subunit and the 10' residue of the adjacent subunit. In all GlyR -subunits the 10' residue is a threonine, but in the -subunit it is a serine. The second intersubunit contact occurs between residues homologous to Q14' and T13' in neighboring TM2s of the GlyR 1-subunit (or E14' and S13' in the -subunit).
Although the 19' position defines the external border of membrane-embedded portion TM2, the -helical structure extends into extracellular space for another 2.5 turns before terminating at the residue corresponding to V280 in the 1-GlyR (267). SCAM analyses on the GlyR and GABAAR confirm that most extramembranous TM2 residues have extensive contact with water (37, 234). In fact, the SCAM analysis on the GABAAR even predicted an -helical structure for these residues (37). The TM2-TM3 linker is formed by the 1-subunit residues, V280 to D284.
The roles of TM2 in forming the channel gate, in controlling ionic selectivity, and in forming the binding sites for agents of physiological and pharmacological importance are considered in sections IV and VI.
According to the Miyazawa TM domain structure, the 1-GlyR TM3 -helical domain starts at I285, with the membrane-embedded portion extending from A288 to H311 (267). There is strong support from functionally based techniques that at least the external (NH2-terminal) half of this domain forms an amphipathic -helix. For example, evidence from the nAChR using lipophilic probes identified an -helical like periodicity in the lipid-facing residues (40). A tryptophan scanning analysis identified an identical periodicity in the same set of residues (76). In addition, SCAM analysis of the GABAAR using water-soluble reagents also supported an -helical periodicity in the outer half of TM3 (400, 401). A satisfying aspect of these studies was that the water-facing residues were generally displaced by one from the lipid-facing residues (see Ref. 223 for review). Consistent with structural predictions (267), the SCAM results strongly suggest that this portion of the domain contributes to the lining of a water-filled pocket distinct from the channel pore. Recent SCAM studies on the GABAAR in the absence and presence of pharmacological agents suggest that this pocket changes conformation as the channel gates (400–402). An abundance of evidence from both the GABAAR and GlyR, reviewed in section VI, provide a strong case that residues in this pocket form binding sites for alcohols and volatile anesthetics. The Miyazawa TM domain structure reveals that residues from TM3 are closely apposed to residues from both TM2 and TM4 at several points throughout their lengths (267). However, TM3 appears to contact TM1 only towards the intracellular membrane surface.
In addition to direct structural evidence (267), several lines of functional evidence imply that this domain forms an -helix throughout its entire length. First, the pattern of lipid-exposed residues is consistent with an -helical periodicity as determined by both hydrophobic probes in the nAChR (40, 41) and tryptophan scanning mutagenesis in the GABAAR (171). Second, proteolytic studies on GlyR 1-homomers did not identify cleavages in membrane-associated fragments of this domain (221), a result that is also consistent with an -helical structure. By structural homology with the nAChR, the 1-GlyR TM4 is likely to be initiated at K385 and terminated at V418, with the intramembranous portion extending from K389 to I408 (267). Thus the -helix extends about 2.5 turns beyond the external membrane boundary. Although TM4 is closely apposed to TM3 throughout its length, its contact TM1 appears confined to its intracellular half (267).
C. NH2-Terminal Ligand-Binding Domain
1. Structural homology with AChBP
The fresh water snail, Lymnaea stagnalis, produces and stores a soluble AChBP in glial cells located near to cholinergic synapses. When released by acetylcholine stimulation, AChBP buffers the acetylcholine in the synaptic cleft (350). This protein forms a stable homopentamer and binds acetylcholine, 
-tubocurarine, and -bungarotoxin with much the same affinity as does the 7-homomeric nAChR (350). AChBP comprises 210 amino acids and, although it lacks the TM domains, it provides a full-length model of the NH2-terminal ligand-binding domain of LGICs. It also incorporates the signature cysteine loop that is a unique feature of the LGIC family. It shares a 20–24% amino acid sequence homology with nAChR subunits and a 17% homology with the GlyR 1 subunit (Fig. 2). The crystal structure of this protein (52) represents a major breakthrough in our understanding of LGIC structure and function. Due to both its significant sequence homology and to its functional similarity with the 7-homomeric nAChR, its structure is considered an accurate template of the NH2-terminal ligand-binding domain of the nAChR and, by inference, of other LGIC members.
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1-subunit ligand-binding domain based on the AChBP structure is shown in Figure 3. Each of the five subunits is positioned in a radially symmetrical manner around the central pore. When viewed from above (i.e., from the synapse, looking towards the membrane), the protein is said to resemble "a 5-bladed windmill toy" (52). Individual subunits contain an -helix near the NH2-terminal extremity and then a series of 10 -sheets with short 310 helices following the second and third -sheets. The -sheets 1–7 form a "twisted -sandwich" with -sheets 8–10, resulting in 2 separate hydrophobic cores. Together the -sheets form a modified immunoglobulin fold. Pockets are present at the subunit interfaces, approximately midway between the top andbottom of the protein, and abundant evidence (reviewed in Refs. 74, 179) identifies these as ligand binding sites. This pocket is lined by three loops from one subunit that form the "principal" (or +) side of the ligand-binding pocket, whereas three -sheets from the adjacent subunit form the "complementary" (or –) side of the pocket. Viewed from the top of the complex, the complementary side of each AChBP binding site is situated anticlockwise relative to the principal side (52). The AChBP binding site opens to the outside of the complex and, unlike the Torpedo nAChR electron diffraction images (266), there is no entry to the binding site from the central pore side of the protein.
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It was recently proposed that sections of the 1-GlyR NH2-terminal domain between residues 158–165 and 181–191 may be associated with the plasma membrane (223). Because the AChBP domain corresponding to 158–165 is located at a subunit interface well away from the membrane, a direct membrane interaction seems unlikely. However, residues 181–191 indeed lie toward the lowest point of the structure, between -sheets 8 and 9, and thus could conceivably dip into the membrane.
Apart from the direct polypeptide chain linkage between -sheet 10 and TM1, structural and functional evidence suggest 2 likely points of contact between the ligand-binding domain and the transmembrane domain. These regions are the conserved cysteine loop and the loop linking -sheets 1 and 2 of AChBP. This later loop is also known as "loop 2." Both loops have been proposed to interact closely with the TM2-TM3 linker domain (181, 267), and the nature of these proposed interactions is considered in more detail in section VD.
As shown in Figure 2, the GlyR 1-subunit contains a glycosylation consensus site at N38, with other -subunits containing similar sites at the homologous positions. The GlyR 2-subunit contains additional consensus sites at N45 and N76 (199). On the other hand, the -subunit contains consensus sites at N33 and N220 (137). The first suggestion that the 1-subunit may be glycosylated was the finding that mutations to N38 prevented surface expression of functional 1-GlyRs (10, 200). Recently, it was found that glycosylation of 1-subunits is a necessary prerequisite for homomeric receptor assembly and that receptor assembly is required for transit from the endoplasmic reticulum to the Golgi apparatus and subsequently to the cell membrane (140). The question of whether -subunits are glycosylated remains to be addressed.
As the large TM3-TM4 domain is poorly conserved among LGIC members, both in terms of its length and amino acid sequence, it is likely to exhibit considerable structural variation as well. The only structural information to date suggests that the Torpedo nAChR intracellular domains form a hanging gondola-type structure with transverse holes (or "portals") connecting the pore with the cytoplasm (266). Because these portals are approximately the same size as a permeating ion plus its first hydration shell (266), they are ideally suited to influence ion permeation. Indeed, it has recently been shown that the deletion of three positively charged residues in the 5-HT3AR TM3-TM4 domain dramatically increases the pore unitary cation conductance (183), implying that these residues may frame the portals. The homologous region of the GlyR 1-subunit is denoted by gray shading in Figure 2. The GlyR -subunit has an unusually large internal domain, comprising 130 residues, whereas the 1-subunit contains 86 residues (Fig. 2). The intracellular domains of both - and -subunits contain a variety of sites that mediate interactions between the GlyR and cytoplasmic factors. These putative interaction sites are now considered.
Under appropriate conditions, intracellular ubiquitin molecules can covalently attach themselves to specific lysine side chains on the cytoplasmic protein surface. In fact, multiple ubiquitin molecules can attach themselves end to end in a piggyback manner, resulting in a condition termed "polyubiquitination." Ubiquitination or polyubiquitination precipitates the internalization and degradation of many protein types, including surface-expressed 1-GlyRs (55). Following internalization, the ubiquitin molecules induce the 49-kDa GlyR 1-subunit to be proteolytically nicked into a glycosylated (i.e., NH2-terminal) 35-kDa fragment and a 17-kDa COOH-terminal fragment. These fragment sizes are consistent with the ubiquitination domain lying in the large intracellular domain. The TM3-TM4 domain contains a total of 10 lysine residues (Fig. 2), several of which probably need to be individually ubiquitinated before GlyRs can be endocytosed (55). This mechanism is likely to be important in regulating the number of surface-expressed GlyRs per postsynaptic density.
Because prolines induce kinks into peptide chains, regular spacing of these residues can form helical structures known as polyproline (PII) helices. Circular dichroism studies reveal the GlyR 1-subunit to contain a significant fraction (9%) of this structure (58). A certain class of protein-protein interaction sites, termed SH3 domains, are formed from PII helices (290). As recently noted (58), GlyR 1- and -subunits both contain SH3 consensus sequences in their large intracellular domains (Fig. 2). Although the role of these domains has yet to be investigated, they may be involved in GlyR trafficking or cytoskeletal attachment.
The locations of phosphorylation consensus sites in the 1- and -subunits are shown in Figure 2. The evidence that phosphorylation of these sites is able to modulate GlyR function is considered in section VIIA.
This important molecule has long been known to copurify with the native GlyR as a 93-kDa protein (300). Kirsch and Betz (188) showed that it mediates the clustering of GlyRs at postsynaptic sites. Gephyrin interacts with a large and growing number of binding partners, suggestive of a high degree of complexity in the regulation of GlyR clustering. A description of the interactions of gephyrin with molecules other than the GlyR is beyond the scope of this review. Developments in this area are moving rapidly, and recent progress has been covered in several excellent reviews (189, 190, 219). The GlyR gephyrin contact site was isolated to an 18-amino acid domain in the central region of the -subunit TM3-TM4 loop (259) (Fig. 2). Insertion of the gephyrin binding domain into the 1-subunit promotes the clustering of 1-homomeric GlyRs (220, 259). A site-directed mutagenesis study isolated gephyrin bindin
