Laboratorio de Biomembranas, Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina.
J Cell Biochem. 2012 Feb;113(2):580-9. doi: 10.1002/jcb.23382.
We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.
我们之前已经证明，肾皮质集合管细胞（RCCD(1)）在受到低渗应激时，会迅速激活调节性体积减少（RVD）机制。这个过程需要水通道 AQP2 和钙离子内流的存在，这就提出了一个问题，即这种钙离子进入途径的分子身份是什么。由于钙渗透性非选择性阳离子通道 TRPV4 在多种细胞类型中对机械和渗透扰动的反应中起着至关重要的作用，因此本工作的目的是检验以下假设：只有在存在 AQP2 的情况下才会观察到细胞内钙离子浓度的增加和随后的快速 RVD，这可能是由于 TRPV4 的特异性激活。我们评估了 TRPV4 通道的表达和功能及其在 WT-RCCD(1)（不表达水通道蛋白）和 AQP2-RCCD(1)（转染 AQP2）细胞中对 RVD 的贡献。我们的结果表明，这两种细胞系都内源性地表达功能性 TRPV4，然而，只有在表达 AQP2 的细胞中，低渗性才能使通道大量激活。用钌红阻断 TRPV4 会消除钙离子内流和 RVD，从而确定 TRPV4 是体积调节所必需的成分。更重要的是，这个过程依赖于 TRPV4 向质膜的易位。我们的数据提供了 TRPV4 和 AQP2 之间存在新关联的证据，这种关联涉及 TRPV4 被低渗激活和调节细胞对渗透压应激的反应，这表明这两种蛋白组装在一个信号复合物中，以响应非等渗条件。
