Differential subcellular distribution of the α6 subunit versus the α1 and β2/3 subunits of the GABAA/benzodiazepine receptor complex in granule cells of the cerebellar cortex

Baude, Agnès; Séquier, J. M.; McKernan, Ruth M.; Olivier, Kenneth R.; Somogyi, Péter

doi:10.1016/0306-4522(92)90513-2

Cited by 97 publications

(53 citation statements)

References 37 publications

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“…Interestingly, 133 subunit-containing receptors were targeted to the basolateral domain via the apical surface, strongly suggesting that these receptors are capable of transcytosis. Together these results highlight a novel role for receptor 13 subunits in controlling subcellular distribution of GABAA receptors. Therefore, changes in the expression of differing 13 subunit isoforms during development (1, 24) may reflect a requirement for the relocation of GABAA receptors between distinct subcellular locations.…”

Section: Resultsmentioning

confidence: 87%

“…(Bar = 15 ,um. ) cell bodies, and also axons (12)(13)(14)(15). How this differential receptor localization is achieved remains largely unknown.…”

Section: Resultsmentioning

confidence: 99%

“…Collectively, these studies have demonstrated that GABAA receptors can be found on dendrites, cell bodies, and axons (12)(13)(14)(15). To examine the potential role of receptor heterogeneity in controlling the subcellular targeting of GABAA receptors we have used heterologous expression of receptor cDNAs in Madin-Darby canine kidney (MDCK) cells.…”

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confidence: 99%

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Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

Connolly

Wooltorton

Smart

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

130

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y-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: a(1-6), ,3(1-4), y(1-4), and 6(1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the .83 subunit. Expression of a/,8 binary combinations resulted in a nonpolarized distribution for dgl1 complexes, but specific basolateral targeting of both a112 and a1j3 complexes. The polarized distribution of these a/"3 complexes was unaffected by the presence of the y2S subunit. Interestingly, delivery of receptors containing the 133 subunit to the basolateral domain occurs via the apical surface. These results show that 18 subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of ,8-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution ofdifferent 18 subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering. ,y-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. Molecular cloning has revealed that these receptors are members of a ligandgated ion-channel superfamily that includes both nicotinic acetylcholine and glycine receptors (1). Members of this ion-channel superfamily are pentameric hetero-oligomers whose subunits share a common proposed transmembrane topology. This topology comprises a large N-terminal extracellular domain and four transmembrane domains with a large intracellular loop between transmembrane regions 3 and 4 (1). GABAA receptors can be constructed from a range of homologous subunit types, a(1-6), 13(1-4), -y(1-4), and 8(1), creating considerable potential for structural diversity (1). In situ hybridization and immunocytochemical studies suggest a large temporal and spatial diversity of GABAA receptor structure in the brain with many neuron types often expressing multiple receptor subunits (1).Heterologous expression has been used to determine the minimal structural requirements for the production of GABAgated chloride channels. Homomeric subunit expression (e.g., a, 13, or y) has produced various results, with the detection of GABA-gated chloride channels by some groups (2-4), but not by others (5-9). Similarly, expression of binary combinations (e.g., ay, fry) have produced functional GABAA receptors in some cases (10, 11), but not in others (5-8). However, there is general agreement that expression of receptor a and 13 subunits produces GABA-gated channels and that the coexpression of the y2 or y3 subunit is ...

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Section: Resultsmentioning

confidence: 87%

“…(Bar = 15 ,um. ) cell bodies, and also axons (12)(13)(14)(15). How this differential receptor localization is achieved remains largely unknown.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

Connolly

Wooltorton

Smart

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

130

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“…In the adult rat, the granule cell layer of the cerebellum exhibits relatively little [3H]benzodiazepine binding (Sieghart, Eichinger, Richards & Mohler, 1987). This is thought to reflect the high level of expression by granule cells of the a6-subunit (Luiddens et al 1990;Baude et al 1992). When co-expressed together with f,-and y-subunits, the a6-subunit forms atypical receptors that are insensitive to benzodiazepine agonists such as diazepam, but which have a high affinity for the partial inverse agonist Ro 15-4513 (Liiddens et al 1990).…”

Section: Discussionmentioning

confidence: 99%

Whole‐cell and single‐channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

Kaneda

Farrant

Cull-Candy

1995

The Journal of Physiology

222

190

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1. Patch-clamp methods have been used to characterize GABA-and glycine-activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7-to 20-day-old rats. 2. All granule cells responded to 10 M GABA, while -60% responded to 100 /M glycine.With repeated agonist application, whole-cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg-ATP.3. Whole-cell concentration-response curves gave EC50 values of 45-2 and 99-6 /M and Hill slopes of 0 94 and 2-6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole-cell current-voltage (I-V) relationships of GABA-and glycine-activated currents reversed close to the predicted Cl-equilibrium potential. Partial replacement of intracellular Cl-with F shifted the GABA reversal potential to a more negative value. 'Instantaneous' I-V relationships produced by ionophoretic application of GABA were linear, while 'steady-state' I-V relationships produced by ramp changes in potential showed outward rectification. For glycine, 'steady-state' I-V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 uM), SR-95531 (10 /M) and picrotoxinin (100 uM), while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside-out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine-activated singlechannel currents had conductances of 32, 55 and 104 pS. Single-channel I-V relationships were linear.7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 uM) or SR-95531 (10 #M) and were greatly reduced in frequency by tetrodotoxin (ITX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents. 8. In many cells a bicuculline-sensitive 'background current noise' was seen in the presence of glutamate antagonists and TTX. Power spectra of this background noise (following subtraction of noise recorded in the presence of bicuculline) were fitted with the sum of two Lorentzian curves and gave an estimated single-channel conductance of 13-7 pS, comparable to the value of 15-3 pS obtained from spectra of noise produced by externally applied GABA (10 #M).

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“…However, while the ratio of a6 mRNA levels is 1.7 between 7 and 14 DIV, that for the respective translation products expressed in the plasma membrane is 3.6, suggesting either an increase in the translation rate of the mRNA or a decrease in a6 protein metabolism with time in culture. An immunocytochemical electron microscopy study of the distribution in rat cerebellum of GABAA receptors including the a6 receptor subunit showed that these receptors were restricted to postsynaptic sites (granule-cell neurites), whereas the GABAA receptors containing the a1 subunit were present in both synaptic and extrasynaptic locations (neurites and soma) (36). Our previous study of expression of GABAA receptors in cultured cerebellar granule cells (5) showed that at 14 DIV, a6- containing receptors were expressed both in neurites and in soma.…”

Section: Resultsmentioning

confidence: 99%

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

Zheng

Zhu

Puia

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The amounts of mRNAs encoding al, a'6, 132, 133, y2, and 8 subunits of y-aminobutyrate type A (GABAA) receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal rat granule cells in primary culture. We investigated possible correlations (i) between temporal changes in mRNA content and expression density of their respective translation products and (ii) between the quantitative changes of receptor subunit expression, the GABA EC5. for Cl-channel activation, and diazepam efficacy in modulating GABA action on the Clchannels. At 3 days in vitro, the amount of GABAA receptor subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of V2 and 8 subunit expression was smaller than the expression of the al or a6 or 182/133 subunits. This suggests that during in vitro maturation each subunit may be regulated independently and invites speculation as to possible changes in specific GABAA receptor subtype abundance during development in vitro. The maximal current intensity elicited by GABA failed to increase from 5 to 14 days in vitro, though the amount of mRNA encoding various subunits and the expression density of their respective translation products increased. Thus, qualitative changes in the GABAA receptor subtypes expressed and/or abnormalities in the subunit assembly very likely account for the uniformity of the maximal current intensity elicited by GABA during in vitro development. Also, during maturation of neuronal cultures from 5 to 20 days in vitro the extent of the positive modulation of GABA action by diazepam decreased dramatically. This finding might be related to an increase in the abundance of GABAA receptors including the a6 subunit and/or to the expression, during granule cell maturation in vitro, of GABAA receptors devoid of y2 subunits. To study whether changes in GABAA receptor subunit mRNA and protein expression occur synchronously during neuronal maturation in vitro (13) and whether eventual maturational variations in subunit expression could be associated with changes in GABAA receptor function, we used a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique (14-16) to quantify specific GABAA subunit mRNAs, the label-fracture immunogold technique to monitor the expression of GABAA receptor subunits in the plasma membrane of cerebellar granule neurons (4, 5), and whole-cell recordings to assess GABA action and its modulation by diazepam (8). MATERIALS AND METHODSCell Culture. Primary cultures of cerebellar granule cells were prepared from 8-day-old Sprague-Dawley rat pups (13).Cells were dispersed with trypsin and plated at 2 x 10W cells per cm2 on 100-or 35-mm Nunc dishes coated with poly(Dlysine). Cultures were maintained in a humidified 6% CO2 atmosphere at 370C in basal Eagle's medium containing 10%6heat-inactivated fetal bovine serum, 2 mM ...

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Differential subcellular distribution of the α6 subunit versus the α1 and β2/3 subunits of the GABAA/benzodiazepine receptor complex in granule cells of the cerebellar cortex

Cited by 97 publications

References 37 publications

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

Whole‐cell and single‐channel currents activated by GABA and glycine in granule cells of the rat cerebellum.

Changes in gamma-aminobutyrate type A receptor subunit mRNAs, translation product expression, and receptor function during neuronal maturation in vitro.

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