Fig. 4

TRPV4 co-localizes with—and regulates—NKCC1. A Representative immunolabeling in rat choroid plexus of TRPV4 (red) and NKCC1 (green). DAPI is used for nuclei staining. Scale bar = 10 μm. B Proximity ligation assayed interaction complex between NKCC1 and TRPV4 shown as red speckles. Inset: Absence of primary antibodies against TRPV4 and NKCC1. Scale bar = 10 μm. C Efflux of ⁸⁶Rb⁺ from choroid plexus (inset) in control settings without, n = 5 (black) or with NKCC1 inhibition by bumetanide (BUM), n = 5 (grey), or upon TRPV4 activation by GSK without, n = 5 (green) or with BUM, n = 5 (light green). GSK-mediated ⁸⁶Rb⁺ efflux obtained with inclusion of the TRPV4 inhibitor RN, n = 5 (dotted, purple line). Y-axis is the natural logarithm of the amount of left in the choroid plexus at time t (A_t) divided by the amount at time 0 (A₀). D NKCC1-mediated efflux rate constants for ⁸⁶Rb⁺ in control, n = 5 (black) or upon TRPV4 activation, n = 5 (GSK; green). Statistical evaluation with Student’s t-test. ***P < 0.001