Display Settings:

Items per page

Results: 6

1.
Figure 6

Figure 6. TREK1 is activated by GABAB in hippocampal neurons. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

(A) Image of a cultured hippocampal neuron expressing TREK1-PCS and soluble Tomato. (B) Representative current obtained from hippocampal neuron expressing TREK1-PCS. Application of baclofen (60 µM) induces an outward current, which can be reversed upon washout. During the baclofen response the current is modulated by light (ii), indicating that TREK1 contributes to the GABAB receptor triggered current. (C) Zoom-in of the recorded current in top right during periods marked as (i), (ii) and (iii). Illumination at 500 nm (green) and 380 nm (magenta). (D) Representative current obtained from hippocampal neuron in organotypic slice expressing TREK1-PCS. During the baclofen response the current is modulated by light, indicating that TREK1 contributes to the GABAB receptor triggered current under conditions when the hippocampal circuit is intact.

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.
2.
Figure 3

Figure 3. Heteromeric channels formed by TREK1-PCS and WT retain normal regulation by external acidification. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

(A) Representative example of the effect of acidic pH on the light-gated current of TREK1-PCS/WT. Alternating illumination at 500 nm (green) and 380 nm (magenta) reversibly blocks and unblocks, respectively, the constant outward current, both at pH7.4 and pH6.5, but the amplitude of the photo-modulation is smaller at pH6.5. (B) Inhibition by external acidification from pH7.4 to pH 6.5 of the photo-blocked current component of TREK1-PCS/WT heteromeric channels is similar to the inhibition of total WT current under the same condition. Numbers in parentheses above bars indicate number of cells tested.

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.
3.
Figure 5

Figure 5. TREK1 in hippocampal neurons. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

(A) Whole-cell recording from hippocampal neurons expressing TREK1-PCS in dissociated neuronal culture (top) and cultured hippocampal slice (bottom). Representative example of light modulation of the resting membrane potential, hyperpolarizing under 500 nm light (green), when TREK1 channels are unblocked, and depolarizing under 380 nm light (magenta) when the channels are blocked. (B) Average of resting membrane potential measured under 500 nm light (green, unblocked) and 380 nm light (magenta, blocked). (C) Photo-modulation of spontaneous firing of hippocampal neuron expressing TREK1-PCS and labeled with MAQ. (D) Average of the spontaneous firing rate over several minutes (2–4 minutes while alternating 500 nm and 380 nm light for 5 s each). Statistical significance determined with paired t-test.

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.
4.
Figure 4

Figure 4. Mutation of Serine 333 to Aspartate reduces the photoswitchable current and prevents its up-regulation by GiPCR activation. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

The residue S333 has been reported to be a PKA phosphorylation site and mutation to aspartic acid (to mimic the phosphorylated state) has been shown to decrease TREK1 current. (A) Alternating illumination between 380 nm (magenta) and 500 nm (green) reversibly blocks and unblocks constant outward current from TREK1-PCS/WT heterodimer (top) and from TREK1-PCS/TREK1S333D dimer (bottom). (B) Bar graph representing the light-gated current recorded from HEK cells transfected by either TREK1-PCS + TREK1 or TREK1-PCS + TREK1S333D. The S333D mutation decreased the photocurrent. (C) Alternating illumination between 380 nm (magenta) and 500 nm (green) reversibly blocks and unblocks constant outward current from TREK1-PCS/WT heterodimer before (top) and after GABA application (bottom). (D) Bar graph representing the effect of activation of GABAB receptor in HEK cells transfected with GABAB1 and GABAB2 in combination with either TREK1-PCS + TREK1 or TREK1-PCS + TREK1S333D. The S333D mutation prevents regulation of photocurrent by GABAB activation.

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.
5.
Figure 2

Figure 2. Development of a subunit replacement strategy. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

(A) Schematic representation of subunit replacement strategy. Deletion of the TREK1 carboxy-terminal tail (TREK1-PCS, grey) results in retention of the homomeric mutant channel in the endoplasmic reticulum. In contrast, the wildtype homomeric channel (WT, blue) traffics to the plasma membrane. Co-expression of TREK1-PCS with WT produces a heteromeric channel that traffics to the membrane because of the WT subunit and which can be light-gated because of MAQ attachment to the TREK1-PCS. (B–C) Whole-cell recording from HEK293T cell expressing either TREK1-PCS alone (B) or co-expressed with WT (C) and labeled with MAQ. Current was elicited by voltage-ramps (from - 100 to 50 mV, 1s in duration) (left). Alternating illumination at 500 nm (green) and 380 nm (magenta) reversibly blocks and unblocks constant outward current, as seen at different holding potentials (right).

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.
6.
Figure 1

Figure 1. Light-gated TREK1. From: Optical control of endogenous proteins with a photo-switchable conditional subunit reveals a role for TREK1 in GABAB signaling.

(A) (Top) MAQ consists of a maleimide (M), which tethers the photoswitch to a cysteine introduced into the outer portion of the first P-loop of the channel (bottom), a photoisomerable azobenzene (A) linker and a quaternary ammonium (Q) pore blocker. (Bottom) Cartoon showing the membrane topology of TREK1 channel and the different position tested. (B) Schematic representation of light-gated TREK1. MAQ is covalently attached to cysteine (S121C). MAQ blocks the pore in the cis configuration (380 nm light). Exposure to 500 nm light places MAQ in the trans state where the pore is unblocked. (C, D) Whole-cell recording from HEK293T cell expressing TREK1(S121C) and labeled with MAQ. Current was elicited by voltage-ramps (from −100 to 50 mV, 1s in duration) (C). Alternating illumination at 500 nm (green) and 380 nm (magenta) reversibly blocks and unblocks constant outward current, as seen at different holding potentials (D).

Guillaume Sandoz, et al. Neuron. ;74(6):1005-1014.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk