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.

中文翻译：

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TRPV4 依赖性 Ca2+ 流入决定质膜上的胆固醇动态

瞬时受体电位香草酸 4 (TRPV4) 是一种 Ca 渗透性非选择性阳离子通道，其活性由其周围的膜脂（例如胆固醇、磷酸肌醇）控制。 TRPV4 的跨膜区包含胆固醇识别氨基酸共有 (CRAC) 基序及其位于质膜胞质小叶中的反向 (CARC) 基序。 TRPV4 定位于细胞膜穴窝（caveolae），这是质膜上一个富含胆固醇的球状结构域。在这里，我们使用全内反射荧光显微镜通过双色单分子成像可视化活细胞质膜上 TRPV4 和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 在质膜的胞质小叶上标记胆固醇。我们的单分子追踪分析表明，TRPV4 分子与 D4H 可接近的胆固醇分子主要共定位于低流动性膜域，其中两种分子高度聚集。在小凹内部和外部均观察到 TRPV4 和 D4H 可接触胆固醇的共定位。激动剂诱发的 TRPV4 激活显着降低了 TRPV4 和 D4H 可接触胆固醇分子之间的共定位概率和关联率。有趣的是，TRPV4 激活后，质膜上 D4H 可接近的胆固醇分子的颗粒密度降低，而处于快速扩散状态的 D4H 可接近的胆固醇分子增加。将骨骼发育不良相关的 R616Q 突变引入 TRPV4 的 CRAC/CARC 基序中，可减少与胆固醇簇的相互作用，但不能改变 D4H 可接近的胆固醇动力学。从机制上讲，TRPV4 介导的 Ca 内流和 TRPV4 的 C 端钙调蛋白结合位点对于调节质膜 D4H 可接近的胆固醇动力学至关重要。我们提出 TRPV4 通过 Ca 内流操纵胆固醇动态来重塑其周围的质膜环境。