Department of Bioengineering & Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, United States; Department of Bioengineering & Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, United States.
Department of Bioengineering & Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, United States.
Biomaterials. 2014 Mar;35(9):2868-77. doi: 10.1016/j.biomaterials.2013.12.030. Epub 2014 Jan 4.
Canonical transient receptor potential channel 6 (TRPC6) can play an important role in governing how cells perceive the surrounding material environment and regulate Ca(2+) signaling. We have designed a TRPC6 reporter based on fluorescence resonance energy transfer (FRET) to visualize the TRPC6-mediated calcium entry and hence TRPC6 activity in live cells with high spatiotemporal resolutions. In mouse embryonic fibroblasts (MEFs), platelet-derived growth factor BB (PDGF) can activate the TRPC6 reporter, mediated by phospholipase C (PLC). This TRPC6 activation occurred mainly at lipid rafts regions of the plasma membrane because disruption of lipid raft/caveolae by methyl-β-cyclodextrin (MβCD) or the expression of dominant-negative caveolin-1 inhibited the TRPC6 activity. Culturing cells on soft materials or releasing the intracellular tension by ML-7 reduced this PDGF-induced activation of TRPC6 without affecting the PDGF-regulated Src or inositol 1,4,5-trisphosphate (IP3) receptor function, suggesting a specific role of mechanical tension in regulating TRPC6. We further showed that the release of intracellular tension had similar effect on the diffusion coefficients of TRPC6 and a raft marker, confirming a strong coupling between TRPC6 and lipid rafts. Therefore, our results suggest that the TRPC6 activation mainly occurs at lipid rafts, which is regulated by the mechanical cues of surrounding materials.
经典瞬时受体电位通道 6（TRPC6）在调节细胞感知周围物质环境和调节 Ca(2+)信号方面起着重要作用。我们设计了一种基于荧光共振能量转移（FRET）的 TRPC6 报告子，以高时空分辨率可视化活细胞中 TRPC6 介导的钙内流和因此 TRPC6 活性。在小鼠胚胎成纤维细胞（MEFs）中，血小板衍生生长因子 BB（PDGF）可以通过磷脂酶 C（PLC）激活 TRPC6 报告子。这种 TRPC6 激活主要发生在质膜的脂筏区域，因为甲基-β-环糊精（MβCD）破坏脂筏/ caveolae 或表达显性失活的 caveolin-1 抑制了 TRPC6 活性。在软材料上培养细胞或通过 ML-7 释放细胞内张力会降低 PDGF 诱导的 TRPC6 激活，而不影响 PDGF 调节的Src 或肌醇 1,4,5-三磷酸（IP3）受体功能，表明机械张力在调节 TRPC6 中具有特定作用。我们进一步表明，细胞内张力的释放对 TRPC6 和脂筏标记物的扩散系数具有相似的影响，证实了 TRPC6 和脂筏之间的强耦合。因此，我们的结果表明，TRPC6 的激活主要发生在脂筏上，其受到周围材料力学线索的调节。
