Priority effects transcend scales and disciplines in biology.

Although primarily studied through the lens of community ecology, phenomena consistent with priority effects appear to be widespread across many different scenarios spanning a broad range of spatial, temporal, and biological scales. However, communication between these research fields is inconsistent and has resulted in a fragmented co-citation landscape, likely due to the diversity of terms used to refer to priority effects across these fields. We review these related terms, and the biological contexts in which they are used, to facilitate greater cross-disciplinary cohesion in research on priority effects. In breaking down these semantic barriers, we aim to provide a framework to better understand the conditions and mechanisms of priority effects, and their consequences across spatial and temporal scales.

Difficulties in obtaining an HPGe detector for low-level measurements.

All low-level laboratories require HPGe detectors to meet certain technical specifications, some of which are not available from manufacturers prior to purchase. Ensuring an HPGe detector is fit for purpose requires the purchase and installation of a detector in the laboratory, incurring both financial risk and considerable time and effort. We show that the optimal HPGe crystal for low-level laboratories has a diameter matched to the source and a length providing 70% absorption of the gamma-rays of interest.

Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.

The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.

Intracellular trafficking of GABA(A) receptors.

Some of the mechanisms that control the intracellular trafficking of GABA(A) receptors have recently been described. Following the synthesis of alpha, beta, and gamma subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While beta3, beta4, and gamma2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA(A) receptors formed from alpha1beta or alpha1betagamma subunits. At the neuronal surface, native GABA(A) receptor clustering and synaptic targeting require a gamma2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA(A) receptor associated protein (GABARAP), may be necessary for the formation of GABA(A) receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA(A) receptor gamma2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA(A) agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA(A) receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA(A) receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.

Chicken GABA(A) receptor beta4 subunits form robust homomeric GABA-gated channels in Xenopus oocytes.

Chicken GABA(A) receptor beta4L and beta4S subunits were expressed in Xenopus oocytes by cRNA injection. Oocytes expressing either beta4 subunit alone or in combination with the chicken alpha1 subunit were studied using the two-electrode voltage-clamp technique. Both the beta4L and beta4S subunits form homomeric GABA-gated Cl- channels with similar efficiencies. In comparison, oocytes expressing either the chicken alpha1 or beta2S polypeptide show no or barely detectable GABA responses, as reported by others for most single-subunit vertebrate GABA(A) receptors. The GABA-gated currents due to the beta4L-subunit homomer were not affected by the presence of actinomycin D during cRNA expression, indicating that nascent oocyte polypeptides are not required for channel formation. The homomeric beta4L-subunit receptors show high affinity for GABA with an EC50 value of 4.3 +/- 0.4 microM and a Hill coefficient of 1.1 +/- 0.1 (n = 6). In response to GABA application at the EC25 value, currents elicited from the beta4L-subunit receptor are enhanced by 50 microM pentobarbital (110 +/- 10%, n = 3) and 10 microM loreclezole (60 +/- 3%, n = 3), inhibited by 10 microM picrotoxinin (93 +/- 3%, n = 3), but not affected by 1 microM diazepam. These properties are similar to those found for oocytes expressing heteromeric chicken alpha1beta4L and alpha1beta2S receptors. Since the beta subunits of GABA(A) receptors provide essential determinants for receptor assembly and subcellular localization, homomeric beta4-subunit receptors are a useful model system for further study of the structure and function of GABA(A) receptors.

Decreased expression of GABAA receptor alpha6 and beta3 subunits in stargazer mutant mice: a possible role for brain-derived neurotrophic factor in the regulation of cerebellar GABAA receptor expression?

The cerebellar granule cells of the spontaneous recessive mutant mouse strain, stargazer (stg/stg), fail to express brain-derived neurotrophic factor mRNA. This deficit is exclusive to these neurons and is believed to underlie the motor irregularities displayed by stg/stg, though the molecular basis for their phenotype has still to be resolved. Brain-derived neurotrophic factor has been shown to play a role in the postnatal maturation of cerebellar granule cells. Differentiation of these neurons, postnatally, is characterised by a switch in their GABAA receptor subunit expression profile. Notably, the GABAA receptor alpha6 subunit, which is specific to these neurons, becomes detectable at postnatal days 10-14 (P10-14). To determine whether cerebellar GABAA receptor expression has been compromised in stg/stg mice, the expression levels of GABAA receptor alpha1, alpha6, beta2 and beta3 subunits were compared between stg/stg mice and the appropriate wild-type background strain, C57BL/6J (+/+). By quantitative immunoblotting, it was found that the expression of the alpha6 and beta3 subunits was 23+/-8% and 38+/-12% (mean+/-S.E.M., n=6) of control (+/+) levels, respectively. In contrast, the expression of the alpha1 and beta2 subunits was not significantly different from controls, being 116+/-11% and 87+/-24% (mean+/-S.E.M., n=6) of +/+ levels, respectively. Total specific [3H]Ro15-4513 binding activity detected in cerebellar membranes prepared from stg/stg was not significantly different from +/+ mice. However, the benzodiazepine agonist-insensitive subtype of [3H]Ro15-4513 binding activity, a pharmacological motif of alpha6 subunit-containing GABAA receptors, was lower in stg/stg mice relative to the +/+ strain which correlated with the lowered level of alpha6 subunit expression. Thus, we have identified an abnormality in the GABAA receptor profile of stg/stg mutant mice that might underpin its irregular phenotype.

Aberrant expression of GABAA receptor subunits in the tottering mouse: an animal model for absence seizures.

The single-locus mutant mouse tottering (tg) is an established model for absence seizures. We have previously reported an impairment in GABA-induced chloride uptake in tg brain [Tehrani and Barnes, Epilepsy Res. 1995;22:13-21]. In order to determine if this alteration in GABAA receptor function can be related to specific receptor isoforms, we examined the radioligand binding properties of GABAA receptors and the expression of GABAA receptor subunit mRNAs in the cerebral cortex. Saturation binding of [3H]flunitrazepam revealed a significantly lower Kd value in tg cortical tissues (1.77 +/- 0.05 nM) in comparison to that for the background C57BL/6J strain (3.23 +/- 0.23 nM), while the Bmax values were indistinguishable. Biphasic displacement of [3H]flunitrazepam binding by 2-oxoquazepam showed that low affinity binding sites account for 36 +/- 7.6 and 51 +/- 7.5% of the total in control and tg, respectively. The level of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to tg cortical membranes was 73.6 +/- 5.8% of that in controls. Paired measurements by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant differences in the levels of GABAA receptor alpha 1, alpha 3, alpha 5, beta 2, beta 3, gamma 2 or gamma 3 subunit mRNAs between tg and control cortex. However, tg tissues showed elevated levels of alpha 2- and beta 1-subunit mRNAs, representing 256 and 177%, respectively, those of controls. For the tg cortex, the enhanced expression of GABAA receptor alpha 2 and beta 1 subunits correlates with recombinant subtypes known to have low affinity for 2-oxoquazepam and impaired binding of TBPS. These aberrant properties of GABAA receptors could influence the development or propagation of phenotypic seizures in the tottering mouse.

Sequestration of gamma-aminobutyric acidA receptors on clathrin-coated vesicles during chronic benzodiazepine administration in vivo.

Chronic administration of benzodiazepine agonists produces behavioral tolerance. For induction of tolerance, the use-dependent down-regulation of gamma-aminobutyric acidA (GABA[A])/ benzodiazepine receptors is a potential cellular mechanism. We previously identified GABA(A) receptors on clathrin-coated vesicles from rat brain, suggesting that surface receptors can be internalized via endocytosis. To examine a role for coated vesicles in GABA(A) receptor down-regulation in vivo, fractions were obtained from mouse brain microsomes through density centrifugation and treatment with 0.1% Triton X-100. This coated vesicle preparation was enriched in clathrin subunits and clathrin light-chain kinase and had twice the level of [3H]flunitrazepam binding as did vesicles not exposed to Triton. Adult mice were treated with lorazepam (2 mg/kg/day) for 7 days via osmotic minipump, achieving a serum level of 103 +/- 8.9 ng/ml. The level of flunitrazepam bound to coated vesicles was increased by 83 +/- 13% in the lorazepam-treated mice compared with vehicle-treated controls. The Bmax value for [3H]flunitrazepam binding to synaptic membranes from lorazepam-treated animals was 33 +/- 4% lower than that of controls. The amount of GABA(A) receptor alpha-1 subunits, as quantified by Western blotting, followed a similar pattern. Relative to controls, immunoreactivity for alpha-1 subunits in coated vesicles from lorazepam-treated mice was increased by 60.0 +/- 10.3%, whereas that in synaptic membranes declined by 12 +/- 6%. These results indicate that lorazepam-dependent GABA(A) receptor sequestration occurs in mouse brain. Furthermore, it is suggested that this sequestration may play a role in GABA(A) receptor down-regulation in vivo.

Repression of gamma-aminobutyric acid type A receptor alpha1 polypeptide biosynthesis requires chronic agonist exposure.

Although it is well established that the number of gamma-aminobutyric acid type A (GABAA) receptors declines in cortical neurons exposed to GABAA receptor agonists, the mechanisms responsible for this use-dependent down-regulation remain unclear. Two hypotheses have been proposed: (i) agonist-evoked sequestration and degradation of surface GABAA receptors and (ii) repression of receptor subunit biosynthesis. We have addressed this problem using [35S]Met/Cys pulse-chase labeling of chick cortical neurons in culture and immunoprecipitation and immunoblotting with an antibody (RP4) directed against a GABAA receptor alpha1-(331-381) fusion protein. Exposure of the cells to GABA or isoguvacine for 2 h to 4 days had no effect on the initial rate of 35S incorporation into the GABAA receptor 51-kDa alpha1 polypeptide, but this rate declined by 33% after a 7-day treatment. This is consistent with a previous report (Baumgartner, B. J., Harvey, R. J., Darlison, M. G., and Barnes, E. M. (1994) Mol. Brain Res. 26, 9-17) that a 7-day GABA treatment of this preparation produced a 45% reduction in the alpha1 subunit mRNA level, while a 4-day exposure had no detectable effect. On the other hand, after a 4-day exposure to these agonists, a 30% reduction in the level of the alpha1 polypeptide was observed on immunoblots, similar to that found previously for down-regulation of GABAA receptor ligand-binding sites. Thus, the de novo synthesis of GABAA receptor alpha1 subunits is subject to a delayed use-dependent repression that was observed after, rather than before, the decline in neuronal levels of the polypeptide. Pulse-chase experiments showed a monophasic degradation of the GABAA receptor 35S-alpha1 subunit with a t1/2 = 7.7 h, a process that was unaffected by the addition of GABA to neurons during the chase period. These nascent 35S-labeled polypeptides are presumably diluted into the neuronal pool of unlabeled unassembled alpha1 subunits, which was found to exceed by a 4:1 molar ratio the amount assembled into [3H]flunitrazepam binding sites. Thus, the data reveal an alternative scheme for degradation of GABAA receptor polypeptides: a pathway that may participate in the agonist-independent degradation of unassembled receptor subunits. This differs from another pathway for the agonist-dependent degradation of mature GABAA receptors derived from the neuronal surface (Calkin, P. A., and Barnes, E. M., Jr. (1994) J. Biol. Chem. 269, 1548-1553).

Developmental expression of chick cortical GABA(A) receptor alpha1 subunits in vivo and in vitro.

In order to examine the expression of the GABA(A) receptor alpha1 subunit during chick cortical development in vivo and in vitro, we have utilized a polyclonal antibody (RP4) directed against an alpha1(331-381) fusion protein. This antibody exhibits a high titer for precipitation of [3H]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta2- or beta4-subunit fusion proteins, and a robust reaction with a single 51-kDa polypeptide on immunoblots of cortical membranes. This indicates monospecificity of the RP4 antiserum for the GABA(A) receptor alpha1 subunit. The alpha1-subunit antibody also showed strong immunocytochemical reactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum. The ontogeny of the alpha1 subunit in chick cortex and in derived neuronal cultures was examined by quantitative Western blotting. The level of the alpha1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expression at day 4. Expression of the cortical GABA(A) receptor alpha1 subunit increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alpha1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), showed a corresponding incline. These findings correlated well with previous developmental studies of GABA(A) receptor ligand binding sites both in vivo and in vitro. The parallel increase of the alpha1 subunit transcript and polypeptide with [3H]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical ontogeny. This was investigated further by quantitative immunoprecipitation. At saturation, the RP4 antiserum consistently precipitated 50-65% of the central [3H]flunitrazepam binding sites in the developing cortex from E12 through P7, despite a 5-fold increase in the binding level. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha1 subunits remains relatively constant. Furthermore, the alpha1 polypeptide appears to be a major component of GABA(A) receptor oligomers at all stages of cortical maturation.

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors.

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABAA receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [3H]muscimol. a GABAA-specific agonist. [3H]flunitrazepam a benzodiazepine agonist, and [35S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABAA receptor channels. Under standard conditions, the binding level of [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS to coated vesicles represented 12.3 +/- 1.8%, 7.9 +/- 1%, and 10.2 +/- 1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [3H]flunitrazepam binding site with a KD value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [3H]flunitrazepam and [35S]TBPS binding assays. For [3H]flunitrazepam binding to synaptic membranes, GABA gave an EC50 = 2.0 microM and at saturation (100 microM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [3H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [35S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABAA receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABAA receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110 +/- 14% and 29.5 +/- 3.8%, respectively, of the immunoreactivity for GABAA receptor beta 2 and alpha 1 subunits. Thus, in comparison to GABAA receptors on synaptic membranes, those on CCVs have a reduced alpha 1/beta 2-subunit ratio. It may be suggested that a selective decline in the content of alpha 1 subunits in coated vesicles could in part account for GABAA receptor uncoupling.

Use-dependent regulation of GABAA receptors.

Prolonged occupancy of GABAA receptors by ligands, including GABA and benzodiazepine agonists, sets in motion a series of mechanisms that can be termed use-dependent regulation. These mechanisms can be subdivided into two distinct pathways, one for GABAA receptor downregulation and another for upregulation. Treatment of cortical neurons with GABA or benzodiazepines in cultures opens the pathway for GABAA receptor downregulation, which includes (in putative temporal order): (1) desensitization (tachyphylaxis), (2) sequestration (endocytosis) of subunit polypeptides and uncoupling of allosteric interactions between GABA and benzodiazepine binding sites, (3) subunit polypeptide degradation, and (4) repression of subunit gene expression. The end-point of GABAA receptor downregulation, a reduction in receptor number, is postulated to be established initially by degradation of the receptor protein and then maintained by a diminished level of de novo synthesis. Benzodiazepine treatment of many preparations, including cells expressing recombinant GABAA receptors, may elicit only desensitization, sequestration, or uncoupling, without a decline in receptor number. Components of the GABAA receptor downregulation pathway are also evoked by chronic administration of GABAmimetics, benzodiazepines, barbiturates, and neurosteroids in animals. This downregulation correlates with the establishment of tolerance to and physical dependence on the pharmacological effects of these drugs, suggesting a cellular model for this behavior. The upregulation of GABAA receptors is observed as one of the neurotrophic actions of GABA, primarily in cultured cerebellar granule cells. Upregulation in culture is caused by enhanced expression of genes for GABAA receptor subunits and correlates with the establishment of GABAergic circuitry in the developing cerebellum. Thus, both the upregulation and downregulation of GABAA receptors appear to represent use-dependent pathways for guiding synaptic plasticity in the vertebrate central nervous system.

Reduced function of gamma-aminobutyric acidA receptors in tottering mouse brain: role of cAMP-dependent protein kinase.

The single-locus mutant mouse tottering (tg) displays spontaneous seizures that resemble those in human petit-mal epilepsy. In order to examine alterations in GABAA receptor function which could arise as a result of this mutation, the influx of 36Cl- was determined using microsacs (membrane vesicles) isolated from the brain of tg/tg and coisogenic C57BL/6J (+/+) control mice. In microsacs from both tg/tg and +/+ strains, the maximum level of 36Cl- uptake induced by 50 microM GABA was observed during five seconds of incubation at 28 degrees C. Compared to +/+, the GABA-dependent 36Cl- uptake in tg/tg microsacs was significantly lower and faded rapidly during longer incubations. The levels of gated 36Cl- uptake in tg/tg microsacs were 45 +/- 6.3%, 65 +/- 9.9%, and 33 +/- 6.1% of control (+/+) values for 3-, 5-, and 10-s incubations, respectively. GABAA receptor-specific agonists (30 microM), muscimol, isoguvacine and THIP (4,5,6,7-tetrahydroisoazolo-[5,4-c]pyridin-3-ol) induced 36Cl- influx in the order muscimol > GABA > isoguvacine > THIP. This order was similar for both strains, but the agonist-dependent influx was always significantly lower in tg/tg compared to +/+. Treatment of the microsacs with 10 microM H-89, a membrane-permeant inhibitor of the cAMP-dependent protein kinase (protein kinase A, PKA), was without effect on GABA-gated 36Cl- uptake in +/+, but increased the gated uptake in tg/tg microsacs by 44 +/- 16%. PKA was assayed using [gamma-32]ATP and kemptide as the substrate. Triton X-100 (0.1%) increased both the basal and 8-Br-cAMP dependent PKA activity in microsacs by 3-4 four fold, showing that most of the enzyme was intravesicular. In the presence of Triton, the basal activity of PKA in the tg/tg preparations was twice that of +/+, while the strain difference was no longer apparent in assays containing 8-Br-cAMP. The data suggest that an abnormal elevation of protein kinase A activity in tottering mouse brain contributes to an impairment of GABAA receptor function. It is suggested that the resulting loss of inhibition could play a role in induction of the seizures which characterize the mutant phenotype.

Agonist administration in ovo down-regulates cerebellar GABAA receptors in the chick embryo.

Chick embryos with an undeveloped blood-brain barrier were used to examine the down-regulation of GABAA receptors in vivo. The GABAA receptor agonist isoguvacine (5 mumol) was applied to the vascularized chorioallantoic membrane of 8 day embryos. This treatment was repeated on embryonic days 11, 14, and 17, and the embryos were sacrificed on day 18 (stage 42). Isoguvacine administration reduced the clonazepam-displaceable binding of [3H]flunitrazepam to washed cerebellar membranes by 34.0 +/- 3.0% compared to vehicle-treated controls. Binding reductions of lower magnitude were found in membranes from the cerebrum and optic lobes. Administration of isoguvacine had no significant effect on the wet weights of whole embryos or cerebella, the yield of cerebellar membranes, or the binding of [3H]N-methylscopolamine. The reduction of [3H]flunitrazepam binding to cerebellar membranes was dose-dependent, allowing a half saturation value of 8 microM isoguvacine to be estimated. Scatchard analysis showed that the Bmax for [3H]flunitrazepam binding was reduced by 28.3 +/- 6.7% compared to controls, without a change in the Kd. Embryonic exposure to isoguvacine also caused a reduction of 43.6 +/- 6.0% in the binding of the GABAA receptor channel ligand [35S]t-butylbicyclophosphorothionate to washed cerebellar membranes. Taken together, these results indicate that isoguvacine induces a down-regulation of the receptor subunits in vivo. However, measurements of cerebellar GABAA receptor mRNAs for the alpha 1, beta 2L, beta 2S, beta 4, gamma 1, gamma 2L, and gamma 2S subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) revealed no significant alterations by isoguvacine administration. The data suggest that translational or post-translational mechanisms, rather than those modulating the synthesis or stability of subunit mRNAs, take precedence in establishing GABAA receptor down-regulation.

Developmental up-regulation and agonist-dependent down-regulation of GABAA receptor subunit mRNAs in chick cortical neurons.

We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression of GABAA receptor subunit genes in cultured neurons from the chick embryo cerebral cortex. During maturation of the neurons between day 2 and day 8 in culture, levels of the alpha 1 subunit transcript (per ng total RNA) increased 3.8 +/- 0.3 fold, while those for the beta 2S and beta 4S subunits increased 2.4 +/- 0.4 and 1.8 +/- 0.2 fold, respectively. The accumulation of the beta 4 S subunit mRNA was more rapid than those encoding either the alpha 1 or beta 2S polypeptides. After 4 days in culture the beta 4S subunit transcript level reached 105 +/- 7.7% of that found after 8 days, while the corresponding amounts for the alpha 1 and beta 2S subunit mRNAs were 50 +/- 7.1% and 44 +/- 10.7%, respectively. On the other hand, no significant differences were observed in the level of either the gamma 1 or the gamma 2S subunit mRNA during development in vitro. Likewise, the ratios of the large/small splice variants (beta 2 = 0.16 +/- 0.02; beta 4 = 0.57 +/- 0.02; gamma 2 = 0.30 +/- 0.06) did not show detectable changes during this period. To study the down-regulation of the mRNAs, a single dose of 100 microM GABA was added to the culture medium. After 7 days of exposure to GABA, the levels of transcripts for the alpha 1, beta 2, beta 4, gamma 1, and gamma 2 subunits and their splice variants (where present) were all reduced by 47-65% compared to untreated controls.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptors in mouse cortical homogenates are phosphorylated by endogenous protein kinase A.

Biochemical, molecular, and electrophysiological studies suggest that phosphorylation of beta subunits of the GABAA receptor (GaR) by exogenous protein kinase A inactivates the receptor channels. We have developed a method which for the first time allows the study of GaR phosphorylation in brain tissues by endogenous PKA. Desalted homogenates or crude synaptic membranes from mouse cerebral cortex were incubated with [gamma-32P]ATP and 8-Br-cAMP or chlorophenylthio-cAMP. Extracts from these incubations were immunoprecipitated by polyclonal antibodies against native GaR and analyzed by SDS-gel electrophoresis and autoradiography. In both homogenates and membranes, cAMP-dependent incorporation of 32P was observed for a 57-kDa peptide, and to a lesser extent 51- to 53-kDa peptides. Phosphorylation of affinity-purified GaR by the catalytic subunit of PKA also produced a major 57-kDa phosphopeptide and a minor 51-kDa phosphopeptide. Limited digestion by S. aureus V-8 protease of the 57-kDa phosphopeptide from the desalted homogenates or from purified receptors produced a major 32P-labeled fragment of 11 kDa, suggesting that the phosphorylation site is similar to that shown previously to reduce GaR function. The phosphorylation of GaRs in homogenates was time dependent and blocked by H-89 or protein kinase inhibitor 5-24, specific inhibitors of protein kinase A. Prolonged incubations resulted in dephosphorylation of the 57-kDa phosphoprotein by a microcystin-LR sensitive phosphatase. In cortical homogenates the level of cAMP-dependent phosphorylation of the 57-kDa GaR peptide was more than 5 times that obtained with washed synaptic membranes. However, assays of PKA using the heptamer kemptide as substrate showed that the specific activity in the particulate fraction was 57% that of the homogenate. This suggests that GaRs on synaptic membranes are preferentially phosphorylated by a cytoplasmic form of protein kinase A. By comparing the [3H]flunitrazepam-photolabeled 53-kDa GaR subunit with the 51-57 kDa [32P]peptides from cortical homogenates, the molar ratio of [32P]/[3H] was estimated at 0.43, suggesting that a substantial fraction of the GaR pool is phosphorylated under these conditions.

gamma-Aminobutyric acid-A (GABAA) agonists down-regulate GABAA/benzodiazepine receptor polypeptides from the surface of chick cortical neurons.

The impermeant SH-cleavable reagent, 125I-labeled 3,3'-dithiopropionyl 1-sulfosuccinimidyl 1'-glycyltyrosine (DPSgt) and specific immunoprecipitation were used to quantitate gamma-aminobutyric acid-A (GABAA) receptor polypeptides with domains exposed on the surface of chick cortical neurons. The 125I label incorporated into 50- and 53-kDa receptor subunits was completely removed by washing the cells with glutathione (GSH) buffer. A single dose of 100 microns GABA or GABA agonist was added to the neurons, and after 5 days in culture the cells were washed and labeled with 125I-DPSgt. This treatment with GABA or isoguvacine reduced the level of the receptor 125I-labeled subunits by 50-60%, while the reduction by 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol was less than 17%. The subunit down-regulation by agonist was prevented by the GABAA antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R5135). Direct iodination of membranes isolated from treated cells revealed a similar loss of subunits, indicating that the down-regulated polypeptides are not retained intracellularly. Furthermore, the level of intracellular [3H]flunitrazepam binding (per mg of protein) did not change significantly during chronic GABA treatment, while the fraction of intracellular binding rose from 7% to 15% of the total, owing to a decline in surface binding. The fate of the surface subunits during acute agonist exposure was examined by labeling intact neurons with 125I-DPSgt at 0 degrees C, incubating with GABA for 2-4 h at 37 degrees C and then washing with GSH buffer. Of the 50-53-kDa receptor peptides originally on the surface, 16 +/- 2% became protected from GSH during the 2-h GABA treatment. This sequestration was not found in cells incubated without GABA, with GABA + R5135, or with GABA at 0 degree C. A consistently lower level of sequestered subunits was recovered after a 4- versus 2-h GABA treatment at 37 degrees C, suggesting polypeptide degradation. Overall, the results indicate that GABAA receptor sequestration and subsequent degradation play an important role in agonist-dependent down-regulation.

Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain.

To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABAA/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [3H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [3H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [3H]flunitrazepam to coated vesicles yielded a KD value of 21 +/- 4.7 nM and a Bmax value of 184 +/- 28 fmol/mg. The KD value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The Bmax value for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABAA/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.