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PLoS One
2017 Jan 03;121:e0170572. doi: 10.1371/journal.pone.0170572.
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Low Expression in Xenopus Oocytes and Unusual Functional Properties of α1β2γ2 GABAA Receptors with Non-Conventional Subunit Arrangement.
Baur R
,
Sigel E
.
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The major subunit isoform of GABAA receptors is α1β2γ2. The subunits are thought to surround an ion pore with the counterclockwise arrangement α1γ2β2α1β2 as seen from the outside of the neuron. These receptors have two agonist sites and one high affinity drug binding site specific for benzodiazepines. Recently, this receptor was postulated to assume alternative subunit stoichiometries and arrangements resulting in only one agonist site and one or even two sites for benzodiazepines. In order to force a defined subunit arrangement we expressed a combination of triple and dual concatenated subunits. Here we report that these unconventional receptors express only small current amplitudes in Xenopus oocytes. We determined agonist properties and modulation by diazepam of two of these receptors that resulted in currents large enough for a characterization, that is, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2. The first pentamer predicted to have two benzodiazepine binding sites shows similar response to diazepam as the standard receptor. As expected for both receptors with a single predicted agonist site the concentration response curves for GABA were characterized by a Hill coefficient < 1. β2-α1-γ2/β2-γ2 displayed a mM apparent GABA affinity for channel opening instead of the expected μM affinity. Based on their subunit and binding site stoichiometry, that contradicts all previous observations, their unusual functional properties and their very low expression levels in oocytes, we consider it unlikely that these unconventional receptors are expressed in neurons to an appreciable extent.
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28114407
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Fig 1. Consensus subunit arrangement of α1β2γ2 GABAA receptors and three newly proposed receptors with unconventional subunit arrangement.Number of predicted agonist sites for GABA and modulatory sites for benzodiazepines (BZ) is shown for each receptor form. On the top left is the color code for the subunits.
Fig 2. Dual and triple concatenated constructs used for the construction of pentameric receptors were expressed in Xenopus oocytes.Currents were standardized to the amplitude elicited in oocytes expressing non-concatenated α1, β2 and γ2 subunits (non-concatenated receptors). The same parameters are shown for receptors with consensus subunit arrangement (consensus receptors) that were built in three different ways and for three receptors with non-consensus subunit arrangement (non-consensus receptors), one of them built in two different ways. For some receptors, stimulation by 1 μM diazepam is also shown.
Fig 3. Concentration-response curves for β2-α1-γ2, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2 receptors.Receptors were exposed to subsequently higher concentrations of GABA and the elicited current amplitude was determined. Individual curves were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.D., n = 3â5 from two batches of oocytes. A) Original current traces recorded in an oocyte expressing β2-α1-γ2/β2-γ2. B) Averaged Concentration-response curves for β2-α1-γ2, β2-α1-γ2/α1-γ2 and β2-α1-γ2/β2-γ2.
Fig 4. Stimulation of β2-α1-γ2, β2-α1-γ2/α1-γ2, β2-α1-γ2/β2-γ2 and α1/β2/γ2 receptors by diazepam.Receptors were exposed to subsequently higher concentrations of diazepam in combination with an EC2 GABA concentration and the elicited current amplitude was determined. Individual curves were first normalized to the fitted maximal current amplitude and subsequently averaged. Data are expressed as mean ± S.D., n = 3 from two batches of oocytes.
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