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Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Lin WC, Davenport CM, Mourot A, Vytla D, Smith CM, Medeiros KA, Chambers JJ, Kramer RH - ACS Chem. Biol. (2014)

Bottom Line: The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism.In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents.LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

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Thermal relaxation of the tethered MAB-0. (a)A representativecell (with MAB-0 conjugated α1(T125C)β2γ2S) showingthe slow current reduction in darkness after an initial response measuredin 380 nm. [GABA] = 10 μM. (b) Group data (mean ± SEM, n = 5) showing the time course of thermal relaxation, plottedas changes in the normalized light-sensitive current component (definedin panel a) and fitted with a single-exponential decay (red curve).Recordings were carried out in HEK293T cells held at −70 mV.
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fig3: Thermal relaxation of the tethered MAB-0. (a)A representativecell (with MAB-0 conjugated α1(T125C)β2γ2S) showingthe slow current reduction in darkness after an initial response measuredin 380 nm. [GABA] = 10 μM. (b) Group data (mean ± SEM, n = 5) showing the time course of thermal relaxation, plottedas changes in the normalized light-sensitive current component (definedin panel a) and fitted with a single-exponential decay (red curve).Recordings were carried out in HEK293T cells held at −70 mV.

Mentions: Cis-to-trans isomerization ofazobenzenes can be driven by 500 nm light or will occur spontaneouslyin darkness through thermal relaxation (Figure 1b and ref (16)). Therate of thermal relaxation for a cis azobenzene isdependent on its chemical structure and the local environment.16 Thermal relaxation of MAB-0 (tethered to α1(T125C))was measured by a functional assay illustrated in Figure 3. GABA-elicited current was measured in HEK293Tcells every 2 min in darkness after an initial response under 5 sof 380 nm illumination. The current decreased slowly during the darkperiod but was fully restored by a subsequent illumination of 380nm light, suggesting that the observed current reduction arose fromthermal relaxation of cis MAB-0 (Figure 3a). We plotted the normalized light-sensitive currentover time and fitted the data with a single-exponential decay (Figure 3b). The half-life of cis MAB-0was calculated to be 23.6 ± 2.7 min (n = 5)in darkness. The slow thermal relaxation of cis PTLmakes LiGABAR bistable, allowing the receptor to stay in the uninhibitedstate for minutes after a brief pulse of 380 nm light. This featureprovides a way to minimize possible adverse effects of light duringthe course of an experiment.


Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Lin WC, Davenport CM, Mourot A, Vytla D, Smith CM, Medeiros KA, Chambers JJ, Kramer RH - ACS Chem. Biol. (2014)

Thermal relaxation of the tethered MAB-0. (a)A representativecell (with MAB-0 conjugated α1(T125C)β2γ2S) showingthe slow current reduction in darkness after an initial response measuredin 380 nm. [GABA] = 10 μM. (b) Group data (mean ± SEM, n = 5) showing the time course of thermal relaxation, plottedas changes in the normalized light-sensitive current component (definedin panel a) and fitted with a single-exponential decay (red curve).Recordings were carried out in HEK293T cells held at −70 mV.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215903&req=5

fig3: Thermal relaxation of the tethered MAB-0. (a)A representativecell (with MAB-0 conjugated α1(T125C)β2γ2S) showingthe slow current reduction in darkness after an initial response measuredin 380 nm. [GABA] = 10 μM. (b) Group data (mean ± SEM, n = 5) showing the time course of thermal relaxation, plottedas changes in the normalized light-sensitive current component (definedin panel a) and fitted with a single-exponential decay (red curve).Recordings were carried out in HEK293T cells held at −70 mV.
Mentions: Cis-to-trans isomerization ofazobenzenes can be driven by 500 nm light or will occur spontaneouslyin darkness through thermal relaxation (Figure 1b and ref (16)). Therate of thermal relaxation for a cis azobenzene isdependent on its chemical structure and the local environment.16 Thermal relaxation of MAB-0 (tethered to α1(T125C))was measured by a functional assay illustrated in Figure 3. GABA-elicited current was measured in HEK293Tcells every 2 min in darkness after an initial response under 5 sof 380 nm illumination. The current decreased slowly during the darkperiod but was fully restored by a subsequent illumination of 380nm light, suggesting that the observed current reduction arose fromthermal relaxation of cis MAB-0 (Figure 3a). We plotted the normalized light-sensitive currentover time and fitted the data with a single-exponential decay (Figure 3b). The half-life of cis MAB-0was calculated to be 23.6 ± 2.7 min (n = 5)in darkness. The slow thermal relaxation of cis PTLmakes LiGABAR bistable, allowing the receptor to stay in the uninhibitedstate for minutes after a brief pulse of 380 nm light. This featureprovides a way to minimize possible adverse effects of light duringthe course of an experiment.

Bottom Line: The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism.In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents.LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

ABSTRACT
Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

Show MeSH
Related in: MedlinePlus