Figure 3. Effects of Spadin on the TREK-1 channel activity.

(A–B) Whole-cell currents measured in COS-7 transfected cells in presence of potassium blockers (K⁺ blockers, 10 mM tetraethyl ammonium (TEA), 3 mM 4-aminopyridine (4-AP), 50 nM charybdotoxin, 10 µM glibenclamide, 100 nM apamin). Cells were clamped at −80 mV and voltage changes were either applied by ramp from −100 to 50 mV, 1 s in duration (A, B main panel) or by 10mV steps from −100 to 40 mV, 1.5 s in duration (B inset). Currents were recorded after TREK-1 activation by 10 µM arachidonic acid (aa) and aa + propeptide (PE, 500 nM) (A) or aa + Spadin (100 nM) (B). Native currents were recorded in absence (Control) and in presence of spadin (Spadin 100 nM, B). Peptides were applied via the bath medium. (C) Dose-dependent spadin inhibition of TREK-1 currents, IC₅₀ value at 0 mV is of 70.7 nM. Currents were measured in presence of 10 µM aa. (D–E) Native currents recorded in the presence of K⁺ blockers after stimulation by 10 µM of aa on CA3 pyramidal neurons from hippocampus slices in wild-type mice (D) or in kcnk2 deficient mice (kcnk2 ^−/−) (E) in the presence or the absence of spadin (1 µM). Currents were elicited by a ramp from −100 mV to 50 mV. (F) Native currents on β-TC3 cell line in similar experimental conditions as (D–E). (G–H) Effect of spadin on the firing rate of DRN 5-HT neurons. Spadin (10⁻⁵ M in a 100 µl bolus) or its vehicle was i.p. administered. Recordings started 30 min after the injection and were performed for a maximal duration of 210 min thereafter. (G) Main panel: Samples of “descents” performed along the DRN, showing typical integrated firing rate histograms in a vehicle- (left panel) or in a spadin-treated (right panel) animal. Each cluster represents the electrical activity of one neuron, each bar representing the average number of recorded action potentials per 10 s. Insets, examples of action potential waveforms of 5-HT neurons. (H) 5-HT neuron firing activity, calculated on the basis of all the cells recorded within the successive tracks performed along the DRN. Values at the bottom of each column indicate the total number of neurons recorded (n = 4 mice in both groups).