GLYCINE: SYNTHESIS AND UPTAKE
Glycine is the major inhibitory neurotransmitter in the brainstem and spinal cord, where it participates in a variety of motor and sensory functions. Glycine is also present in the forebrain, where it has recently been shown to function as a coagonist at the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. In the latter, context glycine promotes the actions of glutamate, the major excitatory neurotransmitter (for a discussion of glycine's role as a coagonist of the NMDA receptor, see Excitatory Amino Acid Neurotransmission). Thus, glycine subserves both inhibitory and excitatory functions within the CNS.
Glycine is formed from serine by the enzyme serine hydroxymethyltransferase (SHMT). Glycine, like GABA, is released from nerve endings in a Ca2+-dependent fashion. The actions of glycine are terminated primarily by reuptake via Na+/Cl--dependent, high-affinity glycine transporters. The specific uptake of glycine has been demonstrated in the brainstem and spinal cord in regions where there are also high densities of inhibitory glycine receptors.
Recently, two glycine transporters have been cloned and shown to be expressed in the CNS as well as in various peripheral tissues (11, 19). These glycine transporters are members of the large family of Na+/Cl--dependent neurotransmitter transporters, and both share approximately 50% sequence identity with the GABA transporters discussed above. The deduced amino acid sequence of both cDNAs predicts the typical 12 transmembrane domains characteristic of these transporters. The two glycine cloned transporters have been named GLYT-1 and GLYT-2 in the order that they were reported (11). These transporter cDNAs are transcribed from the same gene and are quite similar in their 3¢ nucleotide sequences. They differ in their 5¢ noncoding regions as well as in the first 44 nucleotides of their coding sequence. Expression of GLYT-1 and GLYT-2 yield transporters with similar kinetic and pharmacological properties. Interestingly, however, the distribution of GLYT-1 and GLYT-2 transcripts measured by in situ hybridization are different. GLYT-1 mRNA also closely parallels the distribution of the glycine receptor. These data suggest that GLYT-1 is primarily a glial glycine transporter whereas GLYT-2 is primarily a neuronal transporter. The mapping of both glycine transporter mRNAs, as well as the glycine receptor subunit mRNAs, confirm the importance of this neurotransmitter in the brainstem and spinal cord, but support a more widespread distribution in supraspinal brain regions than was previously suspected.
Inhibitory glycine receptors are blocked by the plant alkaloid strychnine, which was also first used to label glycine receptors in spinal cord membranes (52, 53). Strychnine poisoning results in muscular contractions and tetany as a result of glycinergic disinhibition and overexcitation. Electrophysiological studies primarily carried out in rodent spinal cord neurons have demonstrated that glycine activates Cl- ion conductance (8). Like GABA, this increase in Cl- ion conductance results in a hyperpolarization of the neuronal membrane and an antagonism of other depolarizing stimuli. Other a- and b-amino acids, including b-alanine and taurine, also activate glycine receptors, but with lower potency (6, 8).
The glycine receptor was first successfully solubilized and purified by Betz and colleagues using affinity purification over an affinity matrix derivatized with aminostrychnine (8). The affinity-purified glycine receptor was shown to consist of two polypeptide subunits of approximately 48 kD (a) and 58 kD (
, respectively. Reconstitution of these polypeptide subunits into lipid vesicles resulted in functional receptors, and intramolecular cross-linking experiments suggested that the native glycine receptor is a pentameric structure. Photoaffinity labeling of the glycine receptor with [3H]strychnine revealed that both the strychnine and glycine binding sites are located on the 48-kD a subunit. Purification of the a-and b-receptor subunits was followed closely by their molecular cloning (7).
The deduced amino acid sequences of the a- and b-glycine-receptor subunits predict structures quite homologous to the subunits of other ligand-gated ion channels, including the GABAA receptor (7). Each subunit has four hydrophobic membrane-spanning sequences, and each shares considerable sequence identity with the other. Several glycine-receptor a-subunit variants have now been identified (a1–4), and, not surprisingly, they differ in their pharmacological properties and level of expression. As mentioned, both the agonist and antagonist binding sites are located on the a subunit, but at different amino acids (50). Interestingly, glycine receptors comprised of a1 subunits are efficiently gated by taurine and b-alanine, whereas a2-containing receptors are not (8). The a1 and a2 genes are expressed in the adult and neonatal brain, respectively. Interestingly, the b-subunit transcript is expressed at relatively high levels in the cerebral cortex and cerebellum, where no a transcripts or specific [3H]strychnine binding sites have been observed. Coexpression of b subunits with a subunits (as opposed to homo-oligomeric a-subunit glycine receptors) results in glycine receptors with pharmacological properties quite similar to native glycine receptors. Nonetheless, the widespread distribution of b-subunit mRNA in brain suggests that other, perhaps strychnine-insensitive glycine receptor isoforms will be found.
Recently, the expression of a1 and a2 subunits has been shown to be developmentally regulated with a switch from the neonatal a2 subunit (strychnine-insensitive) to the adult a1 form (strychnine-sensitive) at about 2 weeks postnatally in the mouse (8). The timing of this "switch" corresponds with the development of spasticity in the mutant spastic mouse (5), prompting speculation that insufficient expression of the adult isoform may underlie some forms of spasticity.
A convergence of scientific effort—involving molecular pharmacologists, molecular biologists, and medicinal chemists—has revealed a remarkable and, for the most part, unsuspected degree of complexity and heterogeneity in the biosynthetic enzymes, transporters, and receptors for GABA and glycine. For the neuropsychopharmacologist, GABA and glycine-containing and receptive neurons are of particular significance because they are among the best-characterized of all drug targets. Many psychoactive drugs which alter (increase or decrease) CNS excitability do so by effecting GABAergic or glycinergic neurotransmission. Some of these drugs (e.g., benzodiazepine and nonbenzodiazepine anxiolytic–hypnotics) are commonly prescribed for a variety of disorders. It is likely that the wealth of new information on GABA and glycine will result in an even better understanding of their potential role(s) in various neuropsychiatric disorders and in the discovery even more of effective therapeutic agents.
GABA and Glycine
Steven M. Paul