Division of Physiological Chemistry and Metabolism, Graduate School of Pharmaceutical Sciences, Keio University, Tokyo, Japan; Cellular Informatics Laboratory, RIKEN Cluster for Pioneering Research (CPR), Saitama, Japan.
Cellular Informatics Laboratory, RIKEN Cluster for Pioneering Research (CPR), Saitama, Japan; Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.
Biophys J. 2024 Apr 2;123(7):867-884. doi: 10.1016/j.bpj.2024.02.030. Epub 2024 Mar 2.
The activities of the transient receptor potential vanilloid 4 (TRPV4), a Ca-permeable nonselective 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. To this aim, we labeled 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 molecules was decreased and the D4H-accessible cholesterol molecules in the fast-diffusing state were increased at the plasma membrane. The introduction of skeletal dysplasia-associated R616Q mutation into the CRAC/CARC motif of TRPV4, which reduced the interaction with cholesterol clusters, could not alter the D4H-accessible cholesterol dynamics. Mechanistically, TRPV4-mediated Ca influx and the C-terminal calmodulin-binding site of TRPV4 are essential for modulating the plasmalemmal D4H-accessible cholesterol dynamics. We propose that TRPV4 remodels its surrounding plasmalemmal environment by manipulating cholesterol dynamics through Ca influx.
瞬时受体电位香草酸亚型 4（TRPV4）是一种 Ca 通透性非选择性阳离子通道，其活性受周围膜脂（如胆固醇、磷酸肌醇）的调节。TRPV4 的跨膜区含有胆固醇识别氨基酸基序（CRAC）和位于质膜胞质小叶的反式（CARC）基序。TRPV4 定位于质膜的 caveolae，这是一种富含胆固醇的泡状结构域。在此，我们通过全内反射荧光显微镜的双色单分子成像，在活细胞中可视化 TRPV4 与质膜胆固醇之间的时空相互作用。为此，我们使用胆固醇生物传感器 D4H 标记质膜胞质小叶的胆固醇。我们的单分子追踪分析表明，TRPV4 分子与 D4H 可及胆固醇分子主要在低流动性膜域中发生共定位，在这些膜域中，两种分子高度聚集。在 caveolae 内外都观察到 TRPV4 和 D4H 可及胆固醇的共定位。激动剂诱导的 TRPV4 激活显著降低了 TRPV4 与 D4H 可及胆固醇分子之间的共定位概率和缔合速率。有趣的是，在 TRPV4 激活后，质膜上 D4H 可及胆固醇分子的颗粒密度降低，快速扩散状态的 D4H 可及胆固醇分子增加。将与胆固醇簇相互作用减少的骨架发育不良相关的 R616Q 突变引入 TRPV4 的 CRAC/CARC 基序中，不会改变 D4H 可及胆固醇的动力学。在机制上，TRPV4 通过 Ca 内流和 TRPV4 的 C 端钙调蛋白结合位点调节质膜上 D4H 可及胆固醇的动力学，从而重塑其周围的质膜环境。我们提出 TRPV4 通过操纵胆固醇动力学来重塑其周围的质膜环境。
