Fig. 1: Traction force and shear stress activate GABA_B receptor.

a Schematic illustration depicting the modulation of GABA_B receptor activity by ligands (e.g. agonists: GABA and baclofen; antagonist: CGP54626). b Schematic representation of the experiments applying traction force and shear stress to cells in this study, encompassing conditions such as cell suspension or adhesion; PDL or FN coating; and shear stress treatment. No GABA was added in these experiments. c IP₁ production in HEK293 cells transfected with vector or GABA_B receptor, along with G_qi9 chimera in the corresponding conditions: suspension or adhesion (left); PDL or FN coating (middle), without (control) or with shear stress (15 dyn/cm², 15 min) (right). Data are present as mean ± s.e.m. from at least three biologically independent experiments (from left to right, n=4, 5, 5, respectively), each performed in triplicate and analyzed using an unpaired t-test (two-tailed) to determine significance. ***P < 0.001, *P < 0.05, not significant (ns) > 0.05. d Real-time recording of intracellular Ca²⁺ release in HEK293 cells expressing GABA_B receptor and G_qi9. Left: Schematic presentation of shear stress loading device and real-time recording of Ca²⁺ response in single cells. Right: Real-time Ca²⁺ signal measurement. After the recording of basal state of Ca²⁺ release for 50 seconds, cells were subjected to shear stress for 100 seconds. Shear stress was then halted for 150 seconds, after which baclofen was injected and the Ca²⁺ signal was measured for another 200 seconds. Data are present as mean ± s.e.m. from 85 cells recorded. e Traction force and GABA-induced G_i protein activation in HEK293 cells measured by optimized G_i protein BRET sensors. Schematic presentation of Gi protein activation measurement by BRET assay based on G_αi1-Nluc, Venus-G_γ2 and endogenous G_β rearrangement (left). Traction force- (middle) and GABA- (right) induced G_i protein activation in HEK293 cells transfected with vector or GABA_B receptor together with G_i protein sensor. Cells were treated under suspension or adhesion conditions. The traction force-induced net BRET (middle) was calculated by the BRET ratio obtained under suspension conditions, subtracting the BRET ratio in adhesion conditions. PBS or GABA-induced net BRET (right) was calculated by the BRET ratio before PBS or GABA treatment, subtracting the BRET ratio after PBS or GABA treatment. Data are present as mean ± s.e.m. from five biologically independent experiments, and analyzed using an unpaired t-test (two-tailed) to determine significance. ****P < 0.0001, **P < 0.01.