Figure 1.

Inhibition of distinct activation pathways in Magneto2.0. (a) Schematic depicting independent pathways by which TRPV4 responds to stimuli. pBPB inhibits the PLA2-dependent mechanical response of TRPV4 (24). We refer to this condition as (−)Mech. The mutation Y555A/S556A inhibits the thermal response of TRPV4 (24). We refer to this condition as (−)Therm. (b) Calcium-sensitive fluorescence imaging shows that (−)Mech (and not (−)Therm) has reduced sensitivity to hypoosmotic stimulation compared to wild-type Magneto2.0 (non-transfected (NT), n = 6 separate cell cultures with a total of 3473 cells; Magneto2.0, n = 6 separate cell cultures with a total of 660 cells; (−)Mech, n = 7 separate cell cultures with a total of 711 cells; (−)Therm, n = 5 separate cell cultures with a total of 338 cells). (c) Calcium-fluorescence imaging shows that (−)Therm has a significantly reduced response to thermal stimulation (40°C perfusion) compared to (−)Mech and Magneto2.0. The brief decrease in fluorescence observed upon perfusion of warm buffer is due to the heat deformation of the coverslip that results in a change in focus (NT, n = 3 separate cell cultures with a total of 1482 cells; Magneto2.0, n = 5 separate cell cultures with a total of 395 cells; (−)Mech, n = 4 separate cell cultures with a total of 578 cells; (−)Therm, n = 6 separate cell cultures with a total of 343 cells). Bold lines in ΔF/F₀ versus time represent mean values, and shaded regions represent mean ± standard error (SE) based on the number of independent cell cultures recorded. Bar plots represent the maximal ΔF/F₀ normalized to the maximal ΔF/F₀ for NT cells. The error bars show the mean ± SE of the ratios, calculated from the standard deviation (SD) of the maximal averages used in each ratio and based on the number of independent cell cultures recorded. Except for NT, data are obtained only from mCherry⁺ (transfected) cells. The significances are assessed with a two-tailed unpaired Student’s t-test, (^∗p < 0.05, ^∗∗p < 0.01).