TRPV4-dependent Ca < sup > 2+ < /sup > influx determines cholesterol dynamics at the plasma membrane

Biophys J. 2024 Mar 2:S0006-3495(24)00164-4. doi: 10.1016/j.bpj.2024.02.030. Online ahead of print.ABSTRACTThe activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable non-selective cation channel, are controlled by its surrounding membrane lipids (e.g., cholesterol, phosphoinositides). The transmembrane region of TRPV4 contains a cholesterol recognition amino acid consensus (CRAC) motif and its inverted (CARC) motif located in the plasmalemmal cytosolic leaflet. TRPV4 localizes in caveolae, a bulb-shaped cholesterol-rich domain at the plasma membrane. Here, we visualized the spatiotemporal interactions between TRPV4 and cholesterol at the plasma membrane in living cells by dual-color single-molecule imaging using total internal reflection fluorescence microscopy (TIRFM). To this aim, we labelled cholesterol at the cytosolic leaflets of the plasma membrane using a cholesterol biosensor, D4H. Our single-molecule tracking analysis showed that the TRPV4 molecules colocalize with D4H-accessible cholesterol molecules mainly in the low fluidity membrane domains in which both molecules are highly-clustered. Colocalization of TRPV4 and D4H-accessible cholesterol was observed both inside and outside of caveolae. Agonist-evoked TRPV4 activation remarkably decreased colocalization probability and association rate between TRPV4 and D4H-accessible cholesterol molecules. Interestingly, upon TRPV4 activation, the particle density of D4H-accessible cholesterol mol...
Source: Biophysical Journal - Category: Physics Authors: Source Type: research