SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity.

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.

N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free intracellular calcium.

N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.

Characterization of glutamate binding sites in receptors assembled from transfected NMDA receptor subunits.

Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using L-[3H]glutamate and [3H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg2+ can alter [3H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [3H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [3H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.

Interactions between ifenprodil and the NR2B subunit of the N-methyl-D-aspartate receptor.

Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which demonstrates a 140-fold preference for NR2B over NR2A subunits, although the molecular basis for this subunit specificity is unknown. We have made chimeric receptors by fusing the murine forms of NR2A (epsilon 1) and NR2B (epsilon 2) to localize the high affinity determinants of ifenprodil inhibition on the 2B subunit. Binding experiments with 125I-MK-801 implicated the region between amino acids 198 and 356 of NR2B for high affinity ifenprodil interaction. Site-directed mutants at Arg-337 showed that this residue is absolutely required for high affinity ifenprodil inhibition. Polyamines also modulate the NMDA receptor with a preference for NR2B subunits, and the pharmacology of these agents overlaps with ifenprodil. Although the determinants of the polyamine enhancement of iodo-MK-801 binding also localize to the NH2 terminus of NR2B, the point mutants at Arg-337 form receptors that are polyamine-stimulated at wild type levels. In addition, polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit.

Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1).

Several subtypes of sodium-dependent high affinity (SDHA) glutamate transporters have been pharmacologically differentiated in brain tissue. Recently, four distinct cDNAs (EAAC1, GLT1, GLAST, and EAAT4) encoding Na+-dependent glutamate transporters have been isolated, but the properties of some of these transporters do not fully match the properties of transport observed in brain tissue or astrocyte-enriched cultures. The purpose of the current investigation was to determine whether the pharmacological properties of EAAC1 parallel those observed in cortical or cerebellar synaptosomes, C6 glioma, or primary astrocyte-enriched cultures. EAAC1 cRNA was expressed in Xenopus oocytes, an expression system with no detectable endogenous Na+-dependent glutamate transport activity. EAAC1-mediated glutamate transport was >98% Na+ dependent, and the transport was saturable and consistent with a single site. Glutamate transport activates in EAAC1-injected oocytes and C6 glioma have similar Km values for glutamate (Km = 15-24 microM) and Na+ (apparent Km = 35-50mM), and these values markedly differ from those observed in rat synaptosomes (glutamate, Km = 1-5 microM; Na+, Km = 13-20 mM). Several excitatory amino acid analogues were tested as inhibitors of L-[3H] glutamate transport in oocytes expressing EAAC1 cRNA. The potencies of several compounds for inhibition of EAAC1-mediated transport differed from those previously observed in cerebellar synaptosomes and astrocyte-enriched cultures. Although EAAC1-mediated transport and cortical synaptosomal transport have similar pharmacological profiles, five excitatory amino acid analogues were > or= 3-fold more potent as inhibitors of transport into cortical synaptosomes than of transport into EAAC1-injected oocytes. For example, L-trans-pyrrolidine-2,4-dicarboxylate was approximately 5-fold more potent in cortical synaptosomes, and dihydrokainate was approximately 10-fold more potent in cortical synaptosomes than in EAAC1-injected oocytes. In contrast, all of the compounds examined inhibit transport observed in C6 glioma wtih potencies similar to that observed in oocytes injected with EAAC1 cRNA. Consistent with these data, C6 glioma expressed EAAC1- but not GLT1- and GLAST-like immunoreactivity. Although this immunoreactivity migrated as proteins of slightly different molecular masses in each system, treatment with N-glycosidase F shifted all proteins to a molecular mass consistent with that predicted from the cDNA sequence. In cortical synaptosomes, EAAC1-, GLT1-, and GLAST-like immunoreactives were apparent. These results indicate that (i) EAAC1 but not GLAST or GLT1 transporters are expressed in C6 glioma, (ii) synaptosomes contain a heterogeneous population of transporters, (iii) EAAC1 does not account for the pharmacology previously observed in cortical synaptosomes, and (iv) based on the pharmacology and tissue distribution of EAAC1, GLT1, GLAST, and EAAT4, it appears that there are additional glutamate transporter subtypes.

Modulation of the GABAA receptor by propofol is independent of the gamma subunit.

Many anxiolytics, anticonvulsants and general anesthetics modulate gamma-aminobutyric acid type A (GABAA) receptors. The anxiolytic benzodiazepines potentiate the actions of GABA, and this only at GABAA receptors with gamma subunits. The general anesthetics both potentiate GABA and activate GABAA receptors directly, but their binding sites on the receptor are poorly defined. We examined whether the gamma 2 subunit was required for the modulation of GABAA receptors by the general anesthetic 2,6-diisopropylphenol (propofol). Using the patch-clamp technique, we recorded membrane currents from HEK293 cells transfected with human alpha 2, beta 1 and gamma 2 cDNAs and with alpha 2 and beta 1 cDNAs alone. Both forms of the receptor were activated by GABA and by propofol at low concentrations. At maximal doses, propofol was considerably less effective than GABA as an activator of alpha 2 beta 1 GABAA receptors, but it had an efficacy similar to that of GABA as an activator of alpha 2 beta 1 gamma 2s receptors. In addition to activating currents directly, propofol potentiated currents elicited by GABA recorded from cells expressing either subunit combination. We conclude that the gamma 2 subunit is not a prerequisite for activation of GABAA receptors by propofol or for its potentiation of GABA-activated currents. However, the subunit may contribute to the efficacy of propofol as a GABAA receptor activator.

Transfection of N-methyl-D-aspartate receptors in a nonneuronal cell line leads to cell death.

Neurons grown in culture die when they are exposed to high concentrations (0.1-1 mM) of the neurotransmitter L-glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N-methyl-D-aspartate (NMDA) receptors and the consequent Ca2+ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca2+ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca2+ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca2+ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.

Pharmacological characterization of heterodimeric NMDA receptors composed of NR 1a and 2B subunits: differences with receptors formed from NR 1a and 2A.

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.

Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain.

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.

Biphasic modulation of the strychnine-sensitive glycine receptor by Zn2+.

The effects of extracellular applications of Zn2+ ions on the strychnine-sensitive glycine receptor were studied in cultured rat spinal cord neurons and with recombinant glycine receptors expressed in human embryonic kidney 293 cells. Nanomolar concentrations of Zn2+ enhanced the chloride ion current in response to brief applications of 100 microM glycine. The enhancement of glycine responses increased from 20 nM to 1 microM Zn2+. Higher concentrations of Zn2+ caused a reversal of the potentiation, followed by progressive inhibition of the glycine response up to approximately 20-50 microM Zn2+. The biphasic modulation by Zn2+ appeared essentially identical in native and recombinant glycine receptors. Biphasic Zn2+ modulation was observed both with picrotoxin-insensitive heteromeric (alpha 2/beta) receptors and with picrotoxin-sensitive homomeric receptors consisting only of alpha 2 subunits. This suggests that the alpha subunit alone is sufficient for formation of two distinct Zn2+ binding sites on the glycine receptor. The demonstration of Zn2+ modulation of the strychnine-sensitive glycine receptor is of potential physiological importance, in view of the likely range of subsynaptic Zn2+ concentrations to which the receptor is exposed.

Molecular and pharmacological characterization of GABAA receptors in the rat pituitary.

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. alpha 1, beta 1, beta 2, beta 3, and gamma 2s were found to be the predominant subunits in the anterior lobe, whereas alpha 2, alpha 3, beta 1, beta 3, gamma 2s, and gamma 1 were the predominant subunits expressed in the neurointermediate lobe. alpha 5, alpha 6, and delta 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.

Levels of benzodiazepine receptor subtypes and GABAA receptor alpha-subunit mRNA do not correlate during development.

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 alpha 1-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 alpha 1-subunit mRNA were higher than the levels of mRNA encoding other alpha subunits at all developmental time points. The low levels of messenger RNA for alpha 2, alpha 3, and alpha 5 subunits are inconsistent with the high levels of type II benzodiazepine binding in the rat cerebellum at birth because these alpha 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 alpha-subunit gene expression.

Sensitivity of the N-methyl-D-aspartate receptor to polyamines is controlled by NR2 subunits.

The endogenous polyamine spermine has multiple effects on the N-methyl-D-aspartate (NMDA) receptor. These include an increase in the magnitude of NMDA-induced whole-cell currents that is seen in the presence of saturating concentrations of glycine ("glycine-independent" stimulation), an increase in the affinity of the receptor for glycine ("glycine-dependent" stimulation), and voltage-dependent inhibition. Although many of the properties of native NMDA receptors are seen with homomeric NR1 receptors expressed in Xenopus oocytes, we have found that the effects of spermine are differentially regulated by NR2 subunits in heteromeric NR1/NR2 receptors. Glycine-independent stimulation by spermine occurred at homomeric NR1A receptors, which lack the amino-terminal insert in NR1, and at heteromeric NR1A/NR2B receptors but not at heteromeric NR1A/NR2A or NR1A/NR2C receptors. Glycine-independent stimulation was not seen at homomeric NR1B receptors, which include the amino-terminal insert in NR1, or at heteromeric receptors containing NR1B. Thus, glycine-independent stimulation by polyamines requires the presence of an NR1 variant, such as NR1A, that lacks the amino-terminal insert, but the manifestation of the stimulatory effect is controlled by the type of NR2 subunit present in a heteromeric complex. Glycine-dependent stimulation was seen at NR1A/NR2A and NR1A/NR2B receptors and may therefore involve a second polyamine binding site distinct from that which produces glycine-independent stimulation. The voltage-dependent inhibitory effect of spermine, which is more pronounced at hyperpolarized membrane potentials, occurred with similar magnitudes at NR1A/NR2A and NR1A/NR2B receptors but was absent at NR1A/NR2C receptors. Thus, NR2 subunits control both the stimulatory and inhibitory effects of spermine at NMDA receptors. Stimulation but not inhibition by spermine was seen at NR1A/NR2B receptors in the presence of extracellular Mg2+. Stimulation, seen in the presence of physiological concentrations of Ca2+ and Mg2+, may be the predominant effect of polyamines at NMDA receptors in the intact nervous system.

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons.

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.

The expression of a NMDA receptor gene in guinea-pig myenteric plexus.

Muscle tension studies of guinea-pig ileum longitudinal muscle-myenteric plexus preparations suggest that the N-methyl-D-aspartate (NMDA) receptor may be present in the enteric nervous system. Therefore, we investigated the expression of a gene for the NMDA receptor in guinea-pig taenia coli. The gene product was amplified using polymerase chain reaction (PCR) and its synthesis localized using in situ hybridization. A NMDA receptor PCR product from the myenteric plexus was demonstrated with nearly identical sequence characteristics to that in the brain. In situ hybridization studies identified myenteric neurons which express NMDA receptor messenger RNA. Demonstration of the genetic expression of the NMDA receptor supports a role for glutamate as a neurotransmitter in the enteric nervous system.

N-methyl-D-aspartate receptors: different subunit requirements for binding of glutamate antagonists, glycine antagonists, and channel-blocking agents.

Expression of the NR-1 subunit in Xenopus oocytes produces channels that respond to glutamate and are blocked by competitive and noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor. Ionic conductances through these channels are increased by coexpression with NR-2 receptor subunits. We have characterized the pharmacological properties of NMDA receptors assembled from combinations of subunits expressed in transfected cells, to determine the minimum subunit requirements for binding of competitive glutamate antagonists, glycine antagonists, and channel-blocking agents, as detected by ligand-binding experiments. Expression of NR-1a alone produced glycine antagonist binding, whereas the combination of NR-1a and NR-2A was needed to produce binding sites for glutamate antagonists and channel-blocking agents. These results suggest that functional NMDA receptors assemble from these subunits. However, differences in the pharmacological effects of NMDA and polyamines show that not all characteristics of native NMDA receptors are reproduced by this combination of subunits.

Developmental changes in human gamma-aminobutyric acidA receptor subunit composition.

gamma-Aminobutyric acid (GABA) is the neurotransmitter at most inhibitory synapses in the human central nervous system. The GABAA receptor, a ligand-gated ion channel, is the site of action of benzodiazepines, the most widely prescribed neuroactive drugs. It was recently demonstrated that there are multiple subtypes of GABAA receptors. Studies of rodents have shown that receptor subunits are developmentally controlled. The major alpha subunit of the adult receptor is expressed at low levels before birth. This study, using postmortem human tissue, shows that GABAA receptors are present in significant numbers in the human cerebellum at birth, and the numbers rise threefold by adulthood. Two subtypes of benzodiazepine receptors were detected by binding studies in the neonate, whereas only a single subtype of receptor was detected in the adult cerebellum. Comparison to recombinant human GABAA receptors shows that receptors containing alpha 1 constitute 50% of the receptors at birth and the percentage rises to over 95% by adulthood. In both cerebral cortex and cerebellum, a dramatic rise in alpha 1 messenger RNA was observed during development, suggesting that the complement of GABAA receptors differs in infants and adults. These findings have significant implications for normal neurodevelopment as well as for the understanding and treatment of pathophysiological states such as seizures.

Positive modulation of human gamma-aminobutyric acid type A and glycine receptors by the inhalation anesthetic isoflurane.

The interactions of the inhalation anesthetic agent isoflurane with ligand-gated chloride channels were studied using transient expression of recombinant human receptors in a mammalian cell line. Isoflurane enhanced gamma-aminobutyric acid (GABA)-activated chloride currents in cells that expressed heteromeric GABAA receptors consisting of combinations of alpha 1 or alpha 2, beta 1, and gamma 2 subunits and in cells that expressed receptors consisting of combinations of only alpha and beta subunits. Receptors consisting of alpha 2 and gamma 2 subunits were poorly expressed but were sensitive to isoflurane. Receptors consisting of beta 1 and gamma 2 subunits were not expressed. Isoflurane also enhanced glycine-activated chloride currents through homomeric alpha glycine receptors but did not enhance GABA currents in cells expressing homomeric rho 1 receptors. These results show that not all ligand-gated chloride channel receptors are sensitive to isoflurane and, therefore, that the anesthetic interacts with specific structural determinants of these ion channel proteins.