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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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Characterizations forthe α1(T125C)-based LiGABAR. (a) Structure-activityinvestigation of the PTL modules. Photosensitivity of each conjugatedα1(T125C)β2γ2S was indexed at 10 μM GABA. n = 3–7. (b) A representative docking pose of trans MAB-0 (spheres) in a homology model of α1(T125C)β2complex. A positional constraint was applied to mimic the tetheringof trans MAB-0 at α1(T125C) (orange). Residuesof the aromatic box (α1Phe64, β2Tyr97, β2Tyr157,and β2Tyr205) are shown as yellow sticks. (c) Dose-responsecurves for the wild-type α1β2γ2S (black) and MAB-0conjugated α1(T125C)β2γ2S under 380 nm (purple)and 500 nm (green) illumination. n = 3 for the wild-typeand 4 for the conjugated receptor. Data are presented as mean ±SEM. Recordings were carried out in HEK293T cells held at −70mV.
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fig2: Characterizations forthe α1(T125C)-based LiGABAR. (a) Structure-activityinvestigation of the PTL modules. Photosensitivity of each conjugatedα1(T125C)β2γ2S was indexed at 10 μM GABA. n = 3–7. (b) A representative docking pose of trans MAB-0 (spheres) in a homology model of α1(T125C)β2complex. A positional constraint was applied to mimic the tetheringof trans MAB-0 at α1(T125C) (orange). Residuesof the aromatic box (α1Phe64, β2Tyr97, β2Tyr157,and β2Tyr205) are shown as yellow sticks. (c) Dose-responsecurves for the wild-type α1β2γ2S (black) and MAB-0conjugated α1(T125C)β2γ2S under 380 nm (purple)and 500 nm (green) illumination. n = 3 for the wild-typeand 4 for the conjugated receptor. Data are presented as mean ±SEM. Recordings were carried out in HEK293T cells held at −70mV.

Mentions: We next focused on α1(T125C) and investigatedthe structuralrequirements for a PTL to operate at this conjugation site. We changedthe ligand and spacer moieties of MAM-6 and indexed the photoregulationeffects in HEK293T cells (at 10 μM GABA; Figure 2a). The negative charge of muscimol (at physiological pH)is involved in receptor binding. Consistent with this mechanism, replacingmuscimol with a neutral analogue 4-hydroxybenzylamine reduced thephotoregulation effect (I380/I500 = 2.5 ± 0.3 and 1.7 ± 0.1, n = 6 and 3 for MAM-6 and MAB-6, respectively; Figure 2a). Removing the 6-carbon spacer of MAM-6 suppressed photoregulationnearly completely (MAM-0; I380/I500 = 1.3 ± 0.1, n = 4;Figure 2a), suggesting that this spacer isimportant for delivering muscimol into the GABA-binding pocket. Surprisingly,substituting muscimol with 4-hydroxybenzylamine in MAM-0 boosted photoregulation,giving an effect greater than that by MAM-6 (MAB-0; I380/I500 = 3.4 ± 0.4, n = 7; Figure 2a).


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)

Characterizations forthe α1(T125C)-based LiGABAR. (a) Structure-activityinvestigation of the PTL modules. Photosensitivity of each conjugatedα1(T125C)β2γ2S was indexed at 10 μM GABA. n = 3–7. (b) A representative docking pose of trans MAB-0 (spheres) in a homology model of α1(T125C)β2complex. A positional constraint was applied to mimic the tetheringof trans MAB-0 at α1(T125C) (orange). Residuesof the aromatic box (α1Phe64, β2Tyr97, β2Tyr157,and β2Tyr205) are shown as yellow sticks. (c) Dose-responsecurves for the wild-type α1β2γ2S (black) and MAB-0conjugated α1(T125C)β2γ2S under 380 nm (purple)and 500 nm (green) illumination. n = 3 for the wild-typeand 4 for the conjugated receptor. Data are presented as mean ±SEM. Recordings were carried out in HEK293T cells held at −70mV.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Characterizations forthe α1(T125C)-based LiGABAR. (a) Structure-activityinvestigation of the PTL modules. Photosensitivity of each conjugatedα1(T125C)β2γ2S was indexed at 10 μM GABA. n = 3–7. (b) A representative docking pose of trans MAB-0 (spheres) in a homology model of α1(T125C)β2complex. A positional constraint was applied to mimic the tetheringof trans MAB-0 at α1(T125C) (orange). Residuesof the aromatic box (α1Phe64, β2Tyr97, β2Tyr157,and β2Tyr205) are shown as yellow sticks. (c) Dose-responsecurves for the wild-type α1β2γ2S (black) and MAB-0conjugated α1(T125C)β2γ2S under 380 nm (purple)and 500 nm (green) illumination. n = 3 for the wild-typeand 4 for the conjugated receptor. Data are presented as mean ±SEM. Recordings were carried out in HEK293T cells held at −70mV.
Mentions: We next focused on α1(T125C) and investigatedthe structuralrequirements for a PTL to operate at this conjugation site. We changedthe ligand and spacer moieties of MAM-6 and indexed the photoregulationeffects in HEK293T cells (at 10 μM GABA; Figure 2a). The negative charge of muscimol (at physiological pH)is involved in receptor binding. Consistent with this mechanism, replacingmuscimol with a neutral analogue 4-hydroxybenzylamine reduced thephotoregulation effect (I380/I500 = 2.5 ± 0.3 and 1.7 ± 0.1, n = 6 and 3 for MAM-6 and MAB-6, respectively; Figure 2a). Removing the 6-carbon spacer of MAM-6 suppressed photoregulationnearly completely (MAM-0; I380/I500 = 1.3 ± 0.1, n = 4;Figure 2a), suggesting that this spacer isimportant for delivering muscimol into the GABA-binding pocket. Surprisingly,substituting muscimol with 4-hydroxybenzylamine in MAM-0 boosted photoregulation,giving an effect greater than that by MAM-6 (MAB-0; I380/I500 = 3.4 ± 0.4, n = 7; Figure 2a).

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