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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
.
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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.
Figure 1. Engineering of the light-regulated GABAA receptor (LiGABAR).
(a) A LiGABAR is generated by conjugating a photoswitchable tethered
ligand (PTL) onto a receptor comprising the cysteine-substituted α-subunits
(top). In the case of photoswitchable antagonism (bottom), the installed
PTL reversibly isomerizes between two states in response to two different
wavelengths of light, with one preventing and the other enabling GABA
binding (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 α1
subunit. (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 docked
muscimol (red). (d) Representative traces showing reversible photoregulation
of GABA-elicited currents by the tethered MAM-6. Mutant = α1(S68C).
(e) Photoregulation of mutant receptors after MAM-6 conjugation. Each
mutant was coexpressed with the wild-type β2 in Xenopus oocytes. The photoregulation index (mean ± SEM) was measured
at 3 μM GABA, −80 mV. A ratio of 1 indicates no photosensitivity
of the tested receptor.
Figure 2. Characterizations for
the α1(T125C)-based LiGABAR. (a) Structure-activity
investigation of the PTL modules. Photosensitivity of each conjugated
α1(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)β2
complex. A positional constraint was applied to mimic the tethering
of trans MAB-0 at α1(T125C) (orange). Residues
of the aromatic box (α1Phe64, β2Tyr97, β2Tyr157,
and β2Tyr205) are shown as yellow sticks. (c) Dose-response
curves for the wild-type α1β2γ2S (black) and MAB-0
conjugated α1(T125C)β2γ2S under 380 nm (purple)
and 500 nm (green) illumination. n = 3 for the wild-type
and 4 for the conjugated receptor. Data are presented as mean ±
SEM. Recordings were carried out in HEK293T cells held at −70
mV.
Figure 3. Thermal relaxation of the tethered MAB-0. (a)
A representative
cell (with MAB-0 conjugated α1(T125C)β2γ2S) showing
the slow current reduction in darkness after an initial response measured
in 380 nm. [GABA] = 10 μM. (b) Group data (mean ± SEM, n = 5) showing the time course of thermal relaxation, plotted
as changes in the normalized light-sensitive current component (defined
in panel a) and fitted with a single-exponential decay (red curve).
Recordings were carried out in HEK293T cells held at −70 mV.
Figure 4. α1-LiGABAR enables photocontrol over miniature inhibitory
postsynaptic currents (mIPSCs) and epileptiform formation in hippocampal
neurons. (a) A representative continuous trace from a cultured hippocampal
neuron containing α1-LiGABAR. The cell was held at −60
mV and was treated with inhibitors of voltage-gated sodium channels
and ionotropic glutamate receptors. (b) Average mIPSC traces from
the same cell shown in panel a. The green and purple traces represent
average mIPSCs when MAB-0 was in the trans (500 nm)
and cis (380 nm + dark) configuration, respectively.
(c) Quantification of mIPSC photoregulation (mean ± SEM) as the
percent decrease when MAB-0 was switched from cis to trans. The total charge transfer was measured
by integrating the area of average mIPSC.17 (d) Photocontrol over neuronal excitation in a hippocampal slice.
Current-clamp recording was carried out in a LiGABAR-containing CA1
pyramidal neuron. Illumination by 500 nm light resulted in an “epileptic”
plateau potential that was subsequently eliminated by 380 nm light.
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