The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36

CD36 is an 88-kD glycoprotein involved in the cytoadherence of Plasmodium falciparum-parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36.     

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.     

Stereoselective effects of etomidate optical isomers on gamma-aminobutyric acid type A receptors and animals

BACKGROUND: The intravenous anesthetic etomidate is optically active and exists in two mirror-image enantiomeric forms. However, although the R(+) isomer is used as a clinical anesthetic, quantitative information on the relative potencies of the R(+) and S(-) isomers is lacking. These data could be used to test the relevance of putative molecular targets. METHODS: The anesthetic concentrations for a half-maximal effect (EC50) needed to induce a loss of righting reflex in tadpoles (Rana temporaria) were determined for both etomidate enantiomers. The effects of the isomers on gamma-aminobutyric acid (GABA)-induced currents in stably transfected mouse fibroblast cells was also investigated using the patch-clamp technique. In addition, the effects of the isomers on a lipid chain-melting phase transition were determined. RESULTS: The EC50 concentrations for general anesthesia for the R(+) and S(-) isomers were 3.4 +/- 0.1 microM and 57 +/- 1 microM, with slopes of n = 1.9 +/- 0.1 and n = 2.9 +/- 0.2, respectively. The R(+) isomer was also much more effective than the S(-) isomer at potentiating GABA-induced currents, although the degree of stereoselectivity varied with anesthetic concentration. R(+) etomidate potentiated the GABA-induced currents by increasing the apparent affinity of GABA for its receptor. Both isomers were equally effective at disrupting lipid bilayers. CONCLUSIONS: These data are consistent with the idea that the GABA(A) receptor plays a central role in the actions of etomidate. Etomidate exerts its effects on the receptor by binding directly to a specific site or sites on the protein and allosterically enhancing the apparent affinity of GABA for its receptor.     

Differential subunit dependence of the actions of the general anesthetics alphaxalone and etomidate at gamma-aminobutyric acid type A receptors expressed in Xenopus laevis oocytes

Atrial electrical activities during hypothermic, K(+)-induced cardioplegic arrest correlate with an increased incidence of postoperative supraventricular dysrhythmias in coronary artery bypass graft patients. Surface electrocardiogram (ECG) (S-ECG) may be insufficiently sensitive to detect such activity intraoperatively, and invasive methods are impractical and traumatic. From induction of anesthesia until the end of surgery, esophageal ECG signals were detected with a new bipolar esophageal probe and a new high-resolution preamplifier (frequency range 0.01-2000 Hz). The S-ECG and the esophageal ECG (E-ECG) were evaluated independently in 18 patients. Eight of 18 patients presented during cardioplegic arrest a mean of 483 +/- 119 high-amplitude, biphasic P components (mean amplitude 0.7 +/- 0.1 mV, range 0.35-1.15 mV) per patient (mean 36 +/- 6 [5-59] potentials/min) similar to those coinciding with the surface ECG P-waves during sinus rhythm. Six of these eight patients presented a mean of 29 +/- 11 low atrial activities (mean amplitude 0.14 +/- 0.023 mV; range 0.1-0.25 mV) per patient (mean 8.4 +/- 5.6 [2.3-48] potentials/min) in the E-ECG. In the S-ECG, one patient of these eight presented 26 P waves during cardioplegic arrest simultaneously with activities in the E-ECG. During the first 5 days, seven of eight (88%) patients with atrial activities in the E-ECG versus 3 of 10 (30%) patients without atrial activities developed supraventricular tachyarrhythmias postoperatively (P < 0.05). This new high-resolution E-ECG device detected in a beat-to-beat technique more atrial activity during cardioplegic arrest than a S-ECG and offered the advantages of artifact exclusion and better prediction of postoperative supraventricular dysrhythmias.     

Thermodynamics of gamma-aminobutyric acid type A receptor binding differentiate agonists from antagonists

Specific binding of the gamma-aminobutyric acid (GABA)A antagonist [3H]SR 95531 to synaptosomal membranes of rat whole brain was examined between 0 degrees and 37 degrees. Scatchard analysis revealed two (high and low affinity) populations of [3H]SR 95531 binding sites. The Kd values increased with increasing temperature. Ki values for GABAA agonists and antagonists were determined from the displacement of [3H]SR 95531 binding at a low concentration (1.8 nM) of [3H]SR 95531, which binds predominantly to high affinity sites. For most compounds van't Hoff plots (--In Ki, i.e., In Ka, versus 1/T) were linear between 0 degrees and 37 degrees. Curvilinear van't Hoff plots for the antagonists R 5135 and bicuculline methiodide can be attributed to their hydrophobic binding interactions. The enthalpy changes of binding (delta H degrees) were positive for the agonists (muscimol, isoguvacine, GABA, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol hydrochloride, and imidazole-4-acetic acid) and negative for the antagonists (pitrazepin, bicuculline methiodide, R 5135, SR 95531, and SR 95103). Separation of the enthalpic and entropic components of the Gibbs free energy changes of binding (delta G degrees) revealed that binding of the antagonists is driven by both the enthalpic and entropic terms, whereas that of the agonists is driven entirely by entropy changes. A plot of the entropic term (-T delta S degrees) versus the enthalpic term (delta H degrees) showed separate patterns for GABAA agonists and antagonists, with the partial agonists [5-(4-piperidyl)isoxazol-3-ol, imidazole-4-acetic acid, and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol hydrochloride] between them. It is proposed that the entropic term is partly determined by a transition from antagonist to agonist conformation of the GABAA binding sites.     

Coexistence of two beta subunit isoforms in the same gamma-aminobutyric acid type A receptor

Three novel subunit-specific antisera to the beta1, beta2, and beta3 subunits of rat gamma-aminobutyric acid type A (GABAA) receptors have been used to study the native receptor in the rat brain. Affinity-purified anti-beta1, anti-beta2, and anti-beta3 antibodies recognized in immunoblots protein bands of 57, 55, and 57 kDa, respectively. Quantitative immunoprecipitation of solubilized GABAA receptors from various rat brain regions showed that the beta2 subunit was the most abundant isoform in cerebellum (in 96% of the GABAA receptors) and cerebral cortex (64%) but that it was the least abundant isoform in hippocampus (44%). The beta3 subunit was found most abundant in hippocampus (64%) followed by cerebral cortex (48%) and cerebellum (33%). The beta1 subunit was present in a very small proportion of the cerebellar GABAA receptors (3%), but it was present in a high proportion of the GABAA receptors from the hippocampus (49%) and cerebral cortex (32%). Quantitative receptor immunoprecipitation or immunopurification followed by immunoblotting experiments have revealed the existence of colocalization of two different beta subunit isoforms in a significant proportion of the brain GABAA receptors. Thus, in the rat cerebral cortex 33% of the GABAA receptors have both beta2 and beta3 subunits, and 19% of the receptors have both beta1 and beta3 subunits. The extent of colocalization of beta subunit isoforms varied among brain regions, being highest in hippocampus and lowest in cerebellum. These and other results taken together suggest that the number of alpha, beta, and gamma subunits (stoichiometry) in the brain GABAA receptor pentamers might not be unique. It might vary depending on receptor type.     

The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).     

Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.     

Area-specific regulation of gamma-aminobutyric acid type A receptor subtypes by thalamic afferents in developing rat neocortex

Targeting and innervation of the cerebral cortex by thalamic afferents is a key event in the specification of cortical areas. The molecular targets of thalamic regulation, however, have remained elusive. We now demonstrate that thalamic afferents regulate the expression of gamma-aminobutyric acid type A (GABAA) receptors in developing rat neocortex, leading to the area-specific expression of receptor subtypes in the primary visual (V1) and somatosensory (S1) areas. Most strikingly, the alpha1- and alpha5-GABAA receptors exhibited a reciprocal expression pattern, which precisely reflected the distribution of thalamocortical afferents at postnatal day 7. Following unilateral lesions at the birth of the thalamic nuclei innervating V1 and S1 (lateral geniculate nucleus and ventrobasal complex, respectively), profound changes in subunit expression were detected 1 week later in the deprived cortical territories (layers III-IV of V1 and S1). The expression of the alpha1 subunit was strongly down-regulated in these layers to a level comparable to that in neighboring areas. Conversely, the alpha5 subunit was up-regulated and areal boundaries were no longer discernible in the lesioned hemisphere. Changes similar to the alpha5 subunit were also seen for the alpha2 and alpha3 subunits. These results indicate that the differential expression of GABAA receptor subtypes in developing neocortex is dependent on thalamic innervation, contributing to the emergence of functionally distinct areas.     

Excitatory amino acid receptor antagonists block the cardiovascular and anxiety responses elicited by gamma-aminobutyric acidA receptor blockade in the basolateral amygdala of rats

Blockade of gamma-aminobutyric acid (GABAA) receptors in the anterior basolateral amygdala (BLA) with bicuculline methiodide results in an increase in heart rate, blood pressure and "anxiety" in rats. Glutamate receptors in the BLA are also reported to be involved in eliciting anxiety responses. The purpose of this study was to investigate the interaction between GABAergic inhibition and glutamatergic excitation in the BLA. Male Wistar rts were implanted with femoral arterial catheters and bilateral chronic microinjection cannulae into the BLA. Each animal was injected with either artificial cerebrospinal fluid (100 nl), bicuculline methiodide (10 pmol/100 nl) or bicuculline methiodide + one dose of an antagonist of either the N-methyl-D-aspartate receptor [AP5 (20 and 100 pmol) and dizocilpine (25 and 125 pmol)] or the non-N-methyl-D-aspartate ionotropic receptor [CNQX (10 and 50 pmol) and GYKI 52466 (50 and 250 pmol)]. Increases in heart rate, blood pressure and "anxiety" (as measured in the social interaction test) observed in rats after bicuculline methiodide injections into the BLA were blocked in a dose dependent manner with the concurrent injections of either N-methyl-D-aspartate or non-N-methyl-D-aspartate antagonists, suggesting that activation of both subtypes of glutamate ionotropic receptors may be necessary for the responses elicited by GABAA receptor blockade in the basolateral amygdala.     

Inhibition of voltage-dependent sodium channels by the anticonvulsant gamma-aminobutyric acid type A receptor modulator, 3-benzyl-3-ethyl-2-piperidinone

3-Benzyl-3-ethyl-2-piperidinone (3-BEP) belongs to a family of compounds that includes alpha- substituted gamma-butyrolactones, gamma-thiobutyrolactones, 2-pyrrolidinones and hexahydro-2H-azepin-2-ones. Many of these drugs exhibit potent in vivo anticonvulsant activity in mice. Previous electrophysiological studies demonstrated that they potentiate gamma-aminobutyric acid- (GABA) mediated chloride currents. This GABAA receptor modulation was thought to be the main mechanism of anticonvulsant activity. We report that 3-BEP also modulates sodium channels. It decreased sodium currents in cultured rat hippocampal neurons in a voltage- and concentration-dependent manner. The drug's apparent affinity increased as neurons were depolarized. At a holding potential of -60 mV, the apparent IC50 was 487 microM. This concentration is comparable to its EC50 for GABAA modulation (575 microM). Current blockade occurred over all activation voltages tested. The steady state inactivation curve was shifted by 600 microM 3-BEP from V50 = -65.3 mV to -72.0 mV, and recovery from inactivation was slowed from tau = 4.9 to 12.8 msec. Sodium current inhibition was not observed for three related compounds, suggesting a degree of chemical specificity for this activity. We conclude that in addition to its known effects on GABAA receptors, 3-BEP modulates sodium channels. Therefore this compound may prevent seizures by both enhancing inhibition and diminishing neuronal excitability.     

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.     

Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.     

A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid

Degradation is one of several factors that may affect the level of accumulation of transgene products in plants. In plants engineered to secrete antimicrobial proteins to the intercellular compartment of leaves, the degenerative activity of proteases residing in leaf intercellular fluid (IF) could be critical to achieving the expected transgene function. We synthesized a structural analogue (MB39) of the antibacterial protein cecropin B and compared the susceptibility of both proteins to degradation in vitro by IF extracted from leaves of various crops. The half-life of the two proteins in the various IF extracts ranged from 3 min to 25.5 h, with the analogue MB39 displaying the longer half-life in IF from nine of 10 species. Overall, the half-life of MB39 averaged 2.9 times greater than that of cecropin B. Analysis of the peptides produced by endopeptidase activity in potato iF indicated that the 5.7-fold lower degradation rate of MB39 was associated with the substitution of valine for methionine at residue 11 of cecropin B. These findings point to the possibility of tailoring antimicrobial protein genes to reduce the rate of protein degradation in a particular target crop.     

Association between the gamma-aminobutyric acid A3 receptor gene and multiple sclerosis

OBJECTIVE: Non-tumour causes of hyperprolactinaemia, including prolactin-elevating drugs, must be excluded. There is a general view that such drugs are unlikely to raise serum PRL above 3000 mU/I, but the literature is confusing. We report 8 patients receiving treatment with neuroleptic drugs, whose serum PRL concentrations were grossly elevated. METHODS: Prolactin was measured using a 2-site immunofluorometric assay (Abbott Laboratories; reference range < 500 mU/l). Seven of the eight women (age range 24-49 years) were symptomatic (galactorrhoea, oligo- or amenorrhoea). RESULTS: Prolactin concentrations ranged from 3600 mU/l to 7300 mU/l. All patients had a normal pituitary CT scan. Five patients were treated with bromocriptine without detriment to their mental state. CONCLUSION: Prolactin can rise to concentrations associated with prolactinomas in patients on neuroleptic drugs. As it is rarely possible to stop the drugs to see if the PRL concentration will decline to normal, neuroradiology is required in these patients to exclude a vision-threatening macroprolactinoma before deciding on medical treatment.     

The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins

To better define the role of accessory protein as mediators of estrogen receptor (ER) function, we have used immuno-, steroid-, and site-specific DNA-affinity chromatography to identify and characterize proteins that associate with ER in extracts from ER-expressing Chinese hamster ovary (CHO-ER) cells. Two associated proteins [70 and 55 kilodaltons (kDa)] were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis silver stain analysis of all three column eluates. Two additional proteins (45 and 48 kDa) were preferentially retained by the ER-specific DNA affinity column. The 70-kDa protein was subsequently identified by Western blot analysis as a heat shock protein (hsp70). The 55-kDa protein was identified by N-terminal microsequencing as a member of the protein disulfide isomerase family. The 48- and 45-kDa proteins remain unidentified. To determine the possible differential effects of estrogen agonists and antagonists on human (h) ER interaction with these proteins, CHO-ER cells were labeled with [35S]methionine in the presence of estradiol, 4-hydroxytamoxifen (OH-Tam; partial agonist/antagonist), or ICI 182,780 (complete antagonist). None of these ligands altered the pattern of associated proteins when hER complexes were isolated by adsorption to the vitellogenin A2 estrogen response element (ERE). However, when hER was isolated by immunoadsorption, a reduction in the level of associated hsp70 was observed following treatment with estradiol or OH-Tam, compared to no treatment or treatment with ICI 182,780. By gel retardation analysis, maximal interaction of affinity-purified hER with ERE occurred in the presence of all four associated proteins. Removal of the 48- and 45-kDa proteins and/or hsp70 resulted in a decrease in hER/ERE association, which could be restored by the addition of purified hsp70 and/or a mixture of the 48- and 45-kDa proteins. The increased stability of the restored complex was due primarily to an increase in the association rate of hER with ERE. These results suggest that accessory proteins may be required for maximal ER interaction with EREs and that estrogens and estrogen antagonists may promote differential retention of hsp70 in the presence or absence of a specific ERE.     

Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant

Native gamma-aminobutyric acid type A (GABAA) receptors containing different beta-subunit variants were identified immunobiochemically with antisera recognizing selectively the beta 1-, beta 2-, or beta 3-subunit. As determined by immunoprecipitation, the beta 2-subunit was present in 55-60% of GABAA receptors, while only minor receptor populations contained the beta 1-subunit (16-18%) or the beta 3-subunit (19-25%). Since the sum of these values amounts to about 100%, it is concluded that GABAA receptors largely contain only a single type of beta-subunit. Pharmacologically, receptors containing the beta 2-subunit differed from those containing the beta 1- or beta 3-subunit by their differential affinities for benzodiazepine receptor ligands. The subunit composition was analyzed biochemically in receptors immunoprecipitated by the beta 2-subunit antiserum. The beta 2-subunit was preferentially associated with the alpha 1-subunit (rarely with the alpha 2-subunit) and with the gamma 2-subunit; negligible or no immunoreactivity was detected for the alpha 3-, alpha 5-, or beta 1-subunit. A stringent co-expression of alpha 1- and beta 2-subunits was confirmed by double immunofluorescence staining on the cellular level. Neurons expressing the beta 3-subunit immunoreactivity were largely double labeled by the alpha 2-subunit antiserum. Thus, the subunit combinations alpha 1 beta 2 gamma 2 and alpha 2 beta 3 gamma 2 represent two main GABAA receptor subtypes, which together amount to 75-85% of the diazepam-sensitive GABAA receptors.     

The differential specificity of chymotrypsin A and B is determined by amino acid 226

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.     

Role of brain allopregnanolone in the plasticity of gamma-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of gamma-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl- uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for gamma2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, and gamma2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5alpha-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl- uptake and the decrease of gamma2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.