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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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Engineering of the light-regulated GABAA receptor (LiGABAR).(a) A LiGABAR is generated by conjugating a photoswitchable tetheredligand (PTL) onto a receptor comprising the cysteine-substituted α-subunits(top). In the case of photoswitchable antagonism (bottom), the installedPTL reversibly isomerizes between two states in response to two differentwavelengths of light, with one preventing and the other enabling GABAbinding (and the subsequent gating of the transmembrane channel).(b) The structure and photochemistry of MAM-6 (the prototype PTL).(c–e) Identification of MAM-6 attachment sites in the α1subunit. (c) Distribution of the tested cysteine-substituted residues(orange; side chain in sticks) in a homology model of α1β2.24 The GABA-binding site is indicated by a dockedmuscimol (red). (d) Representative traces showing reversible photoregulationof GABA-elicited currents by the tethered MAM-6. Mutant = α1(S68C).(e) Photoregulation of mutant receptors after MAM-6 conjugation. Eachmutant was coexpressed with the wild-type β2 in Xenopus oocytes. The photoregulation index (mean ± SEM) was measuredat 3 μM GABA, −80 mV. A ratio of 1 indicates no photosensitivityof the tested receptor.
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fig1: Engineering of the light-regulated GABAA receptor (LiGABAR).(a) A LiGABAR is generated by conjugating a photoswitchable tetheredligand (PTL) onto a receptor comprising the cysteine-substituted α-subunits(top). In the case of photoswitchable antagonism (bottom), the installedPTL reversibly isomerizes between two states in response to two differentwavelengths of light, with one preventing and the other enabling GABAbinding (and the subsequent gating of the transmembrane channel).(b) The structure and photochemistry of MAM-6 (the prototype PTL).(c–e) Identification of MAM-6 attachment sites in the α1subunit. (c) Distribution of the tested cysteine-substituted residues(orange; side chain in sticks) in a homology model of α1β2.24 The GABA-binding site is indicated by a dockedmuscimol (red). (d) Representative traces showing reversible photoregulationof GABA-elicited currents by the tethered MAM-6. Mutant = α1(S68C).(e) Photoregulation of mutant receptors after MAM-6 conjugation. Eachmutant was coexpressed with the wild-type β2 in Xenopus oocytes. The photoregulation index (mean ± SEM) was measuredat 3 μM GABA, −80 mV. A ratio of 1 indicates no photosensitivityof the tested receptor.

Mentions: The GABAARs, a group of neurotransmitter-gatedchloride-permeablechannels, are therapeutic targets in psychiatric disorders3 and epilepsy4 owingto their inhibitory control over neuronal excitation. They are alsotargets for many drugs of abuse, including alcohol, barbiturates,and benzodiazepines.3,5,6 TheGABAARs are heteropentameric assemblies composed of twoα, two β, and one tertiary subunit (usually γ orδ, Figure 1a).3,5 Amongthese components, the α-subunit is key in determining receptorlocalization and gating kinetics3,5,7,8 and, together with the βsubunit, forms the GABA-binding site.3 Thereare six distinct α-isoforms expressed heterogeneously in differentneuron types and brain regions.3,5,6 Adding to this complexity, a neuron can express multiple α-isoformsthat are differentially distributed in subcellular compartments.7 These findings suggest that each α-isoformhas unique roles in neuronal signaling, and understanding their individualfunctions will provide key insights into GABAAR-associateddisorders and therapeutics.


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)

Engineering of the light-regulated GABAA receptor (LiGABAR).(a) A LiGABAR is generated by conjugating a photoswitchable tetheredligand (PTL) onto a receptor comprising the cysteine-substituted α-subunits(top). In the case of photoswitchable antagonism (bottom), the installedPTL reversibly isomerizes between two states in response to two differentwavelengths of light, with one preventing and the other enabling GABAbinding (and the subsequent gating of the transmembrane channel).(b) The structure and photochemistry of MAM-6 (the prototype PTL).(c–e) Identification of MAM-6 attachment sites in the α1subunit. (c) Distribution of the tested cysteine-substituted residues(orange; side chain in sticks) in a homology model of α1β2.24 The GABA-binding site is indicated by a dockedmuscimol (red). (d) Representative traces showing reversible photoregulationof GABA-elicited currents by the tethered MAM-6. Mutant = α1(S68C).(e) Photoregulation of mutant receptors after MAM-6 conjugation. Eachmutant was coexpressed with the wild-type β2 in Xenopus oocytes. The photoregulation index (mean ± SEM) was measuredat 3 μM GABA, −80 mV. A ratio of 1 indicates no photosensitivityof the tested receptor.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Engineering of the light-regulated GABAA receptor (LiGABAR).(a) A LiGABAR is generated by conjugating a photoswitchable tetheredligand (PTL) onto a receptor comprising the cysteine-substituted α-subunits(top). In the case of photoswitchable antagonism (bottom), the installedPTL reversibly isomerizes between two states in response to two differentwavelengths of light, with one preventing and the other enabling GABAbinding (and the subsequent gating of the transmembrane channel).(b) The structure and photochemistry of MAM-6 (the prototype PTL).(c–e) Identification of MAM-6 attachment sites in the α1subunit. (c) Distribution of the tested cysteine-substituted residues(orange; side chain in sticks) in a homology model of α1β2.24 The GABA-binding site is indicated by a dockedmuscimol (red). (d) Representative traces showing reversible photoregulationof GABA-elicited currents by the tethered MAM-6. Mutant = α1(S68C).(e) Photoregulation of mutant receptors after MAM-6 conjugation. Eachmutant was coexpressed with the wild-type β2 in Xenopus oocytes. The photoregulation index (mean ± SEM) was measuredat 3 μM GABA, −80 mV. A ratio of 1 indicates no photosensitivityof the tested receptor.
Mentions: The GABAARs, a group of neurotransmitter-gatedchloride-permeablechannels, are therapeutic targets in psychiatric disorders3 and epilepsy4 owingto their inhibitory control over neuronal excitation. They are alsotargets for many drugs of abuse, including alcohol, barbiturates,and benzodiazepines.3,5,6 TheGABAARs are heteropentameric assemblies composed of twoα, two β, and one tertiary subunit (usually γ orδ, Figure 1a).3,5 Amongthese components, the α-subunit is key in determining receptorlocalization and gating kinetics3,5,7,8 and, together with the βsubunit, forms the GABA-binding site.3 Thereare six distinct α-isoforms expressed heterogeneously in differentneuron types and brain regions.3,5,6 Adding to this complexity, a neuron can express multiple α-isoformsthat are differentially distributed in subcellular compartments.7 These findings suggest that each α-isoformhas unique roles in neuronal signaling, and understanding their individualfunctions will provide key insights into GABAAR-associateddisorders and therapeutics.

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