Dolan B. Pritchett

Neurosteroids act on recombinant human GABAA receptors

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N‐methyl‐d‐aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low‐affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5′ insert and with or without both 3′ inserts. In cerebellum, however, the major NR1 variants were those containing the 5′ insert and lacking both 3′ inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co‐expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high‐affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.

Functional chloride channels by mammalian cell expression of rat glycine receptor subunit

Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.

A steroid recognition site is functionally coupled to an expressed GABAA−benzodiazepine receptor

Pregnane steroids, particularly 3 alpha-hydroxylated metabolites of progesterone, are known to have rapid and profound effects on brain excitability. Recent evidence suggests that the gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor-Cl- ionophore complex may mediate these actions. The data further suggest that these steroids modulate the complex through a novel site independent of other known sites on the complex. The hypothesis that this site is on the GABA(A)-benzodiazepine receptor-Cl- ionophore complex is tested in the present study by determining its presence on transiently expressed GABAA-benzodiazepine receptors.

Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125I‐MK‐801 binding assays and electrophysiological measures. Binding of 125I‐MK‐801 to cells transfected with NR 1a/2B is saturable with a KD of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK‐801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125I‐MK‐801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA‐induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.

A novel subtype of muscarinic receptor identified by homology screening

A new member of the protein superfamily of G-protein coupled receptors has been isolated by homology screening. By virtue of its homology with other muscarinic acetylcholine receptors and its ability to bind muscarinic specific antagonists, this muscarinic receptor subtype is designated M4. The M4 mRNA is preferentially expressed in certain brain regions. The existence of multiple receptor subtypes encoded by distinct genes in the brain has functional implications for the molecular mechanisms underlying information transmission in neuronal networks.

Molecular and Pharmacological Characterization of GABAA Receptors in the Rat Pituitary

Abstract: Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. α1, β1, β2, β3, and γ2s were found to be the predominant subunits in the anterior lobe, whereas α2, α3, β1, β3, γ2s, and γ1 were the predominant subunits expressed in the neurointermediate lobe. α5, α6, and δ subunits were not detectable. Hill and Scatchard analysis of [3H]muscimol binding to anterior and neurointermediate lobe membranes showed high‐affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central‐type benzodiazepine antagonist [3H]Ro 15‐1788 bound to a high‐affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218 872 displacement of Ro 15‐1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue‐specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.

Chapter 5 Molecular pharmacology of NMDA receptors: modulatory role of NR2 subunits

Publisher Summary N -methyl-D-aspartate (NMDA) receptors are ion-channel receptors in which NR2 subunits are large polypeptides, and they are 50–70% homologous to each other and only 15–20% identical to NR1. The NR1 subunit can form functional NMDA receptors when expressed in oocytes. The native NMDA receptors are heterooligomers composed of combinations of NR1 and NR2 subunits. Recent studies have shown that the inclusion of different NR2 subunits in heteromeric NMDA receptors can markedly alter the functional and pharmacological properties of the receptors. Some of these differences are strikingly similar to those seen in the studies of native NMDA receptors in different brain regions or during various stages of development. Receptors containing the NR2C subunit are less sensitive to blockade by Mg 2+ and MK-801 than are receptors containing NR2A or NR2B subunits. Differences in sensitivity to glycine and glutamate-site antagonists have also been reported for receptors containing different NR2 subunits. The consequences of a difference in sensitivity to glycine of native receptors are not known, but if the concentration of glycine in the synapse is not saturating, changes in the concentration of glycine could selectively alter the activity or the activation threshold of some subtypes of NMDA receptors. The inclusion of NR2A in native NMDA receptors is probably responsible for the delayed development of receptors with a low affinity for ifenprodil.

Levels of Benzodiazepine Receptor Subtypes and GABAA Receptor α‐Subunit mRNA Do Not Correlate During Development

Abstract: Developmental changes in the pharmacological properties of the GABAA receptor have been suggested to result from changes in the subunit composition of the receptor complex. The nicotinic acetylcholine receptor is structurally related to the GABAA receptor and undergoes a developmental subunit switch at the neuromuscular synapse. To examine the mechanistic similarities between these systems we sought to find whether the changes in GABAA receptor subunits are controlled by changes in messenger RNA levels, as they are for the nicotinic acetylcholine receptor. We found a 10‐fold increase in the level of α1‐subunit mRNA, and a small increase in levels of GABAA/benzodiazepine receptors from day 1 to day 24 of rat cerebellar development. We also found that the levels of α1‐subunit mRNA were higher than the levels of mRNA encoding other α subunits at all developmental time points. The low levels of messenger RNA for α2, α3, and α5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these α subunits have been shown to form GABAA receptors with type II benzodiazepine binding. These findings are inconsistent with simple models that would explain the developmental differences in GABAA receptor pharmacology simply as a result of changes in α‐subunit gene expression.