Figure 1.
GFP expression and functional h-TREK-1 channel activity on h-TREK-1/HEK cells.
A) Typical pictures of cells observed in transmission and in fluorescence at round three of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. B) Typical pictures of cells observed in transmission and in fluorescence at round fourteenth of cell culture. Functional channel activity was evaluated by current activation with 10 µM AA. C) Real time q-PCR. Levels of TREK-1 or sortlin expression were normalized with the cyclophillin D expression.
Figure 2.
Stretch and pH activation of h-TREK-1 current in cell attached (C.A.) and in inside out (I.O.) patch clamp configurations.
Currents were recorded by application of negative pressure and by internal acidification. A, B) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 5) in cell-attached patch clamp configuration. C, D) Current/pressure relationship and typical current traces obtained on h-TREK-1/HEK cells (closed circle, n = 30) and on HEK cells (closed square, n = 4) in inside-out patch clamp configuration. Inset: negative pressure step protocol, increase −10 mmHg. E, F) Current/potential curves and corresponding histograms obtained after internal acidification on h-TREK-1/HEK cells (n = 8) and on HEK cells (n = 3).
Figure 3.
Pharmacological activation of h-TREK-1 current in whole cell patch clamp configuration.
Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K+ blockers). A) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by AA 10 µM (n = 118). B) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by DHA 10 µM (n = 10). C) Current/potential curves and corresponding current traces obtained before (closed circle) and after (open circle) current activation by ALA 10 µM (n = 10). D) Current/potential curves and corresponding current traces obtained before (closed circle) and after current activation by riluzole 100 µM (n = 12) perfused during 30 s (open circle) or 90 s (closed triangle). Each pharmacological activator was tested on HEK-293 native cells (n = 10) and current/potential curves were shown in the inset of each curve. E) Current density values measured at 0 mV after different times of perfusion of 100 µM riluzole (n = 10 at each time value).
Figure 4.
Pharmacological inhibition of h-TREK-1 current in whole cell patch clamp configuration by spadin (n = 12 for each dose) and fluoxetine (n = 12 for each dose).
Currents were recorded in the presence of a cocktail of potassium channel inhibitors (K+ blockers). The inhibition was obtained after the pre-activation of the current by 10 µM AA. A) Current/potential curves obtained in presence of K+ blockers (closed circles), K+ blockers + AA (open circles) and K+ blockers + AA + spadin 1 µM (closed triangles). The absence of voltage dependence of spadin inhibition (100 nM) was shown in the inset. B) Spadin dose dependent inhibition at 0 mV potential. C) Typical traces of hTREK-1 current pre-activated by 10 µM AA and inhibited by 1 µM spadin. D) Current/potential curves obtained in the presence of K+ blockers (closed circles), K+ blockers + 10 µM AA (open circles) and K+ blockers + 10 µM AA + 30 µM fluoxetine (closed triangles). The absence of voltage dependence of fluoxetine inhibition (10 µM) was shown in the inset. E) Fluoxetine dose-dependent inhibition at 0 mV potential. F) Typical traces of h-TREK-1 current pre-activated by 10 µM AA and inhibited by 30 µM fluoxetine. G, H) Current/potential curves and representative traces of spadin inhibition (1 µM) on 100 µM riluzole activated hTREK-1 current (n = 10). I, J) Current/potential curves and representative traces of fluoxetine inhibition (30 µM) on 100 µM riluzole activated hTREK-1 current (n = 10).
Figure 5.
125I-spadin competition and immunolabeling experiments on h-TREK-1/HEK cells and HEK cells.
A, B) 125I-spadin competition experiments performed on whole cells (n = 3) and on plasma membrane homogenate (n = 3). c) Immunolabelling of h-TREK-1 (green labeling) and sortilin (red labeling) proteins. The co-localization of the two proteins is shown on the Merge picture.
Figure 6.
Oxygen deprivation glucose experiments (OGD) on h-TREK-1/HEK cells and on HEK cells (A, B).
After two hours of OGD in control conditions or with different treatments, survival cells were fixed, labeled and counted (n = 8 for each groups). A) Typical pictures of cells from each tested condition. B) Histograms of number of survival cells (Tukey test, F7,568 = 38.65; *** or ### or $$$ p<0.001) c) The different tested conditions and their corresponding number.