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Sci Rep
2017 Aug 10;71:7770. doi: 10.1038/s41598-017-08031-9.
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Orthosteric- versus allosteric-dependent activation of the GABAA receptor requires numerically distinct subunit level rearrangements.
Amin J
,
Subbarayan MS
.
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Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric α1β2γ2 GABAA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ1 receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA α1β2γ2 receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ1 subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ1 subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABAA ρ1 receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ1 GABAA receptor model system.
Figure 1. Mutations of the 307/328 residues confer sensitivity to the structurally distinct intravenous anaesthetics to the ρ1 receptor. (a) Schematic representation of the ρ1 subunit in the membrane bilayer. The positions of the 307 and 328 residues in the TM2 (II) and TM3 (III) are delineated. (b) Current traces and bar graphs represent the etomidate-dependent potentiation of the ρ1 307/328 mutants. The lines above the current traces show the duration of the drug application. The vertical and horizontal bar scales denote 100 nA and 100 seconds, respectively. (c,d,e,f) The potentiation (as a percent increase) of the EC4 GABA currents in different ρ1 307/328 mutants following the propofol-, ketamine-, midazolam-, and pentobarbital-dependent modulation.
Figure 2. I4AA-, ZAPA-, pentobarbital-, and diazepam-dependent activation of ρ1 307/328 mutants. (a) Pentobarbital (PB)- and diazepam (DZ)-induced current traces in the ρ1, ρI307S/W328I, and ρI307S/W328V receptors. The lines above the current traces represent the duration of the drug application. (b) GABA- and I4AA-evoked current traces in the ρ1, ρI307S/W328I, and ρI307S/W328V receptors. (c) The current maxima of I4AA, ZAPA, PB, and DZ relative to that elicited by GABA in the ρ1, ρI307S/W328I, and ρI307S/W328V receptors. (d) The GABA, I4AA, PB and DZ concentration-response relationships in the ρ1, ρI307S/W328I, and ρI307S/W328V receptors.
Figure 3. Variable co-expression of the ρ1 and 307/328 mutants reveals a distinct activation paradigm for GABA versus pentobarbital. (a) The predicted quantities of the receptor sub-populations resulting from the injection of different ratios of wild-type ρ1 to mutant cRNAs. (b) Current traces represent the maxima of GABA, I4AA, ZAPA, and pentobarbital (PB) in ρ1, ρI307S/W328I, and different ratios of ρ1:ρI307S/W328I. The lines above the current traces represent the duration of the drug application. The vertical and horizontal bar scales represent 100 nA and 100 seconds, respectively. (c) The current maxima of I4AA, ZAPA, and PB relative to that mediated by GABA in ρ1, ρI307S/W328I, and different ratios of ρ1:ρI307S/W328I. The three simulated models are shown in three shades of grey. The model representing the best fit is denoted by a hash # on the bar.
Figure 4. Variable co-expression of the ρ1 and 307/328 mutants demonstrates a distinct activation paradigm for GABA versus diazepam. (a) Current traces represent the maxima of GABA, I4AA, ZAPA, and diazepam (DZ) in ρ1, ρI307S/W328V, and different ratios of ρ1:ρI307S/W328V. The lines above the current traces represent the duration of the drug application. The vertical and horizontal bar scales represent 100 nA and 100 seconds, respectively. (b) The current maxima of I4AA, ZAPA, and DZ relative to that of GABA in ρ1, ρI307S/W328V, and different ratios of ρ1:ρI307S/W328V. The three simulated models are shown in three shades of grey. The model representing the best fit is denoted by a hash # on the bar.
Figure 5. Hetero-oligomeric ρ1 receptors containing a single mutated subunit confer anaesthetic potentiation. (a) The predicted quantities of the receptor sub-populations expected from the co-expression of different ratios of wild-type to mutant cRNA. (b) Current traces elicited by EC4 GABA and EC4 GABA plus 20 or 50 µM PB for ρ1, ρI307S/W328A, and different ratios of ρ1:ρI307S/W328A. The lines above the current traces represent the duration of the drug application. The vertical and horizontal bar scales denote 50 nA and 100 seconds, respectively. (c) PB-dependent potentiation of EC4 GABA for ρ1, ρI307S/W328A, and different ratios of ρ1:ρI307S/W328A. (d) Potentiation of the EC4 GABA by 20, 50, 100, and 200 µM PB for ρ1, and 22:1 ratio of ρ1:ρI307S/W328A as well as ρI307S/W328A (inset). (e) Potentiation of the EC4 GABA by 30 µM DZ for ρ1, and different ratios of ρ1:ρI307S/W328Y. The three shades of grey horizontal lines in c and e are simulated models for the potentiation experiments. The differences in the potentiation levels between the different ratios of the ρ1:mutant and ρ1 are statistically significant (p < 0.05).
Figure 6. A model of GABA- versus anaesthetic-dependent activation. (a) A model of the GABA-dependent activation of α1β2γ2 compared to that of the ρ1 GABAA receptors. T and R represent tense and relaxed states, respectively. Note that in this model, a single GABA binding can stabilize two subunits into a relaxed state and cast a more widespread effect on the overall structure. For the α1β2γ2 receptor, the intermittent binding of two GABA molecules can stabilize four subunits into a relaxed state, while for the ρ1 receptor, three consecutive GABA binding events (three GABA molecules) are needed to achieve the same task. (b) Represents the distinct model of the anaesthetic-dependent activation of the mutated ρ1 receptors. In the presented model, the anaesthetics produce a local and limited effect on the state of the subunits.
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