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The glycine receptor as a paradigm for elegant molecular neuroscience
Neurochem. Int. Vol. 13, No. 2, pp. 147-148, 1988 Printed in Great Britain. All rights reserved
0197-0186/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
CRITIQUE THE GLYCINE RECEPTOR AS A PARADIGM FOR ELEGANT MOLECULAR NEUROSCIENCE SOLOMONH. SNYDER Departments of Neuroscience,Pharmacologyand Molecular Sciences,Psychiatry and BehavioralSciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A.
One of the central questions in neuroscience is how recognition by receptor sites of a neurotransmitter alters cellular function. Molecular cloning of several receptor proteins has provided valuable insights. Most studied are the nicotinic acetylcholine receptor, the p-adrenergic receptor and the GABAbenzodiazepine receptor. By contrast, relatively little attention has been devoted to the inhibitory transmitter glycine. Singlehandedly Heinrich Betz and co-workers have forged a remarkable opus during the past half dozen years characterizing the glycine receptor protein biochemically and, more recently, obtaining its molecular cloning. This work provides a paradigm for elegance in the study of neurotransmitter receptors. For most neurotransmitters in the central nervous system, neurophysiologic evidence came first, with neurochemists arriving later to provide molecular characterization. Glycine may be the first neurotransmitter for which neurochemical investigations have provided the principal insights. High concentrations of endogenous glycine in certain areas of the spinal cord and brainstem suggested that glycine is an inhibitory neurotransmitter of small interneurons (Aprison and Werman, 1965). Werman et al. (1968) showed that synaptic inhibition in the spinal cord involves increased chloride ion conductance and is antagonized by strychnine. Both glycine and GABA increase chloride conductance, but only the effects of glycine are blocked by strychnine. A selective synaptic function of glycine, as opposed to other amino acids, in spinal cord was indicated by a unique sodium dependent high affinity uptake system and depolarization induced release for radiolabeled glycine but not other amino acids in spinal cord and brainstem (Snyder et al., 1973).
Glycine receptors were first labeled in 1973 by reversible ligand binding techniques which, at the time, had only recently been employed to characterize opiate receptors (Young and Snyder, 1973). [3H]strychnine binds with high affinity to sites most concentrated in the spinal cord and brainstem and less in higher centers. Of many neurotransmitters evaluated, glycine and closely related amino acids are most potent with relative affinities for the binding sites paralleling neurophysiologic effects. Evidence that the strychnine binding sites might be linked to the chloride ion channel comes from findings that chloride and other anions influence binding in proportion to their potencies in traversing chloride channels (Young and Snyder, 1974). Biogenic amine, opioid peptide and GABA receptors received much attention in the mid and late 1970s, perhaps in large part because of the important effects of psychotropic drugs upon these sites. Since no major drugs were known to act through glycine receptors, they received less attention until the investigations of Betz. A particularly important early observation was that [3H]strychnine behaves like a photoaffinity agent permitting selective, irreversible labeling of glycine receptors (Graham et al., 1981). Accordingly, glycine receptors could be purified relatively rapidly, much like benzodiazepine receptors photoaffinity labeled with [3H]flunitrazepam (Mohler et al., 1980). The glycine receptor comprises three separate subunits with considerable similarity in their amino acid sequences. A major step forward in understanding the glycine receptor has come with its molecular cloning (Grenningloh et al., 1987). Strategies employed were fairly similar to those used for other receptors. A series of oligonucleotide probes, based on partial 147
148
NOCOMON}]. SNYt)FR
amino acid sequence, permitted identilication ot cDNA clones for the 48 kD subunit. Sequencing of the cDNA revealed substantial homology with the nicotinic acetylcholine receptor and the G A B A benzodiazepine receptor and showed that the chloride channel is part of the glycine receptor protein. One can now divide neurotransmitter receptors roughly into two classes. Receptors that directly open ion channels, such as glycine, GABA and nicotinic acetylcholine receptors, display considerable homology. The other family of receptors with mutual homologies are linked to biochemical second messengers, especially adenylate cyclase, and include rhodopsin, the /3-adrenergic receptor, muscarinic cholinergic receptor and the substance P receptor. The potential clinical and pharmacologic relevance of glycine receptors is becoming more apparent. The disease, familial nonketotic hyperglycinemia, involves motor disorders secondary to increased levels of glycine. The relevance of glycine to the symptoms of the disease is substantiated by the ability of strychnine to alleviate symptoms (Sankaran et al., 1982). The symptoms of hyperglycinemia presumably reflect increased release of glycine from small interneurons which synapse upon motor cells of the spinal cord and brainstem. Absence of glycine neurons or their receptors should result in motor spasticity. Indeed, a genetic mutant mouse, designated spastic, exhibits such symptoms, which in some ways resemble those of individuals suffering from cerebral strokes. These mice display a profound and selective loss of glycine receptors, which apparently accounts for the motor spasticity (White and Heller, 1982). A genetic disorder in cattle, inherited congenital myoclonius, also involves spasticity, myoclonic jerks, and a selective loss of glycine receptors. The recent molecular characterization of glycine receptors and the animal models of motor spasticity attributable to defects in glycine neurotransmission
may serve as a stimulus to drug developmcnl based on glycine. There is a profound need for muscle relaxants in treating many diseases ranging from muscle spasms associated with cervical and lumbar disc disease to the spasticity of children with cerebral palsy and the elderly with cerebrovascular strokes. Benzodiazepines are the most widely used muscle relaxants but cause unacceptable sedation at doses required to elicit muscle relaxation. Benzodiazepines act by facilitating the synaptic effects of GABA, involving presynaptic inhibition of sensory inputs to the spinal cord. Drugs that mimic or facilitate the actions of glycine might offer certain virtues. They would act directly upon motor cells rather than indirectly through sensory afferents. Moreover, since gtycine transmission is prominent only in the spinal cord and brainstem, such drugs may be less likely to cause sedation. Should new therapeutics agents arise from glycine research, they will owe much to the pioneering work of Betz and associates.
REFERENCES
Aprison M. H. and Werman R. (1964) L(fe Sci. 4, 2075-2083. Graham D., Pfeiffer F. and Betz H. (1981 ) Bioehem. biophys. Res. Commun. 102, 1330-1335. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundetfinger E. D. and Betz H. (1987) Nature 328, 215-220. Mohler H., Battersby M. K. and Riehards J. G. (1980) Proc. natn. Aead. Sci. U.S.A. 77, 1666-1670. Sankaran K., Casey R. E., Zaleski W, A. and Mendelson I. M. (1982) Clin. Pediat. 21, 63~637. Snyder S. H., Young A. B., Bennett J. P. and Mulder A. H. (1973) Fed. Proc. 32, 2039-2047. Werman R., Davidoff R. A. and Aprison M. H. (1968) J. Neurophysiol. 31, 81 95. White W. F. and Heller A. H. (1982) Nature 298, 655-4~57. Young A. B. and Snyder S. H. (1973) Proc. natn. Acad. Sci. U.S.A. 70, 2832-2836. Young A. B. and Snyder S. H. (1974) Proc. hath. Acad. Sci. U.S.A. 71, 4002~4005.
0197-0186/88 $3.00 + 0.00 Copyright © 1988 Pergamon Press plc
CRITIQUE THE GLYCINE RECEPTOR AS A PARADIGM FOR ELEGANT MOLECULAR NEUROSCIENCE SOLOMONH. SNYDER Departments of Neuroscience,Pharmacologyand Molecular Sciences,Psychiatry and BehavioralSciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A.
One of the central questions in neuroscience is how recognition by receptor sites of a neurotransmitter alters cellular function. Molecular cloning of several receptor proteins has provided valuable insights. Most studied are the nicotinic acetylcholine receptor, the p-adrenergic receptor and the GABAbenzodiazepine receptor. By contrast, relatively little attention has been devoted to the inhibitory transmitter glycine. Singlehandedly Heinrich Betz and co-workers have forged a remarkable opus during the past half dozen years characterizing the glycine receptor protein biochemically and, more recently, obtaining its molecular cloning. This work provides a paradigm for elegance in the study of neurotransmitter receptors. For most neurotransmitters in the central nervous system, neurophysiologic evidence came first, with neurochemists arriving later to provide molecular characterization. Glycine may be the first neurotransmitter for which neurochemical investigations have provided the principal insights. High concentrations of endogenous glycine in certain areas of the spinal cord and brainstem suggested that glycine is an inhibitory neurotransmitter of small interneurons (Aprison and Werman, 1965). Werman et al. (1968) showed that synaptic inhibition in the spinal cord involves increased chloride ion conductance and is antagonized by strychnine. Both glycine and GABA increase chloride conductance, but only the effects of glycine are blocked by strychnine. A selective synaptic function of glycine, as opposed to other amino acids, in spinal cord was indicated by a unique sodium dependent high affinity uptake system and depolarization induced release for radiolabeled glycine but not other amino acids in spinal cord and brainstem (Snyder et al., 1973).
Glycine receptors were first labeled in 1973 by reversible ligand binding techniques which, at the time, had only recently been employed to characterize opiate receptors (Young and Snyder, 1973). [3H]strychnine binds with high affinity to sites most concentrated in the spinal cord and brainstem and less in higher centers. Of many neurotransmitters evaluated, glycine and closely related amino acids are most potent with relative affinities for the binding sites paralleling neurophysiologic effects. Evidence that the strychnine binding sites might be linked to the chloride ion channel comes from findings that chloride and other anions influence binding in proportion to their potencies in traversing chloride channels (Young and Snyder, 1974). Biogenic amine, opioid peptide and GABA receptors received much attention in the mid and late 1970s, perhaps in large part because of the important effects of psychotropic drugs upon these sites. Since no major drugs were known to act through glycine receptors, they received less attention until the investigations of Betz. A particularly important early observation was that [3H]strychnine behaves like a photoaffinity agent permitting selective, irreversible labeling of glycine receptors (Graham et al., 1981). Accordingly, glycine receptors could be purified relatively rapidly, much like benzodiazepine receptors photoaffinity labeled with [3H]flunitrazepam (Mohler et al., 1980). The glycine receptor comprises three separate subunits with considerable similarity in their amino acid sequences. A major step forward in understanding the glycine receptor has come with its molecular cloning (Grenningloh et al., 1987). Strategies employed were fairly similar to those used for other receptors. A series of oligonucleotide probes, based on partial 147
148
NOCOMON}]. SNYt)FR
amino acid sequence, permitted identilication ot cDNA clones for the 48 kD subunit. Sequencing of the cDNA revealed substantial homology with the nicotinic acetylcholine receptor and the G A B A benzodiazepine receptor and showed that the chloride channel is part of the glycine receptor protein. One can now divide neurotransmitter receptors roughly into two classes. Receptors that directly open ion channels, such as glycine, GABA and nicotinic acetylcholine receptors, display considerable homology. The other family of receptors with mutual homologies are linked to biochemical second messengers, especially adenylate cyclase, and include rhodopsin, the /3-adrenergic receptor, muscarinic cholinergic receptor and the substance P receptor. The potential clinical and pharmacologic relevance of glycine receptors is becoming more apparent. The disease, familial nonketotic hyperglycinemia, involves motor disorders secondary to increased levels of glycine. The relevance of glycine to the symptoms of the disease is substantiated by the ability of strychnine to alleviate symptoms (Sankaran et al., 1982). The symptoms of hyperglycinemia presumably reflect increased release of glycine from small interneurons which synapse upon motor cells of the spinal cord and brainstem. Absence of glycine neurons or their receptors should result in motor spasticity. Indeed, a genetic mutant mouse, designated spastic, exhibits such symptoms, which in some ways resemble those of individuals suffering from cerebral strokes. These mice display a profound and selective loss of glycine receptors, which apparently accounts for the motor spasticity (White and Heller, 1982). A genetic disorder in cattle, inherited congenital myoclonius, also involves spasticity, myoclonic jerks, and a selective loss of glycine receptors. The recent molecular characterization of glycine receptors and the animal models of motor spasticity attributable to defects in glycine neurotransmission
may serve as a stimulus to drug developmcnl based on glycine. There is a profound need for muscle relaxants in treating many diseases ranging from muscle spasms associated with cervical and lumbar disc disease to the spasticity of children with cerebral palsy and the elderly with cerebrovascular strokes. Benzodiazepines are the most widely used muscle relaxants but cause unacceptable sedation at doses required to elicit muscle relaxation. Benzodiazepines act by facilitating the synaptic effects of GABA, involving presynaptic inhibition of sensory inputs to the spinal cord. Drugs that mimic or facilitate the actions of glycine might offer certain virtues. They would act directly upon motor cells rather than indirectly through sensory afferents. Moreover, since gtycine transmission is prominent only in the spinal cord and brainstem, such drugs may be less likely to cause sedation. Should new therapeutics agents arise from glycine research, they will owe much to the pioneering work of Betz and associates.
REFERENCES
Aprison M. H. and Werman R. (1964) L(fe Sci. 4, 2075-2083. Graham D., Pfeiffer F. and Betz H. (1981 ) Bioehem. biophys. Res. Commun. 102, 1330-1335. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundetfinger E. D. and Betz H. (1987) Nature 328, 215-220. Mohler H., Battersby M. K. and Riehards J. G. (1980) Proc. natn. Aead. Sci. U.S.A. 77, 1666-1670. Sankaran K., Casey R. E., Zaleski W, A. and Mendelson I. M. (1982) Clin. Pediat. 21, 63~637. Snyder S. H., Young A. B., Bennett J. P. and Mulder A. H. (1973) Fed. Proc. 32, 2039-2047. Werman R., Davidoff R. A. and Aprison M. H. (1968) J. Neurophysiol. 31, 81 95. White W. F. and Heller A. H. (1982) Nature 298, 655-4~57. Young A. B. and Snyder S. H. (1973) Proc. natn. Acad. Sci. U.S.A. 70, 2832-2836. Young A. B. and Snyder S. H. (1974) Proc. hath. Acad. Sci. U.S.A. 71, 4002~4005.