The role of TRPC6 channels in neutrophil recruitment

中性粒细胞从血液中的募集遵循一系列明确的事件，包括滚动、粘附和迁移，然后是对病变的趋化性。与内皮细胞的牢固粘附是整联蛋白依赖性的，并且由于G蛋白偶联受体（GPCR）对化学引诱物（例如CXCR 2）的活化而发生。CXCR 2像大多数其他GPCR一样含有Na+离子的变构结合位点。Na+结合抑制组成型GPCR活性，其可高达激动剂刺激活性的50%。因此，GPCR内的Na+结合似乎是确保激动剂激活受体的全动态范围的一种方式。CXCR 2配体，分类为“中间趋化物”，驱动嗜中性粒细胞向病变附近的趋化性。“最终目标”化学引诱物，在病变内释放，并典型地由fMLP代表，然后接管。因此，在中性粒细胞募集过程中，化学引诱物以空间上不同的顺序起作用。神经元的募集依赖于Ca 2+。我们已经表明，TRPC 6通道在功能上耦合到CXCR 2依赖的步骤。然而，其他分子定义的Ca 2+内流通道的招聘级联和潜在的机制的各个步骤的贡献还没有得到很好的表征。在这里，我们想要检验以下假设：（i）TRPC 6和TRPM 2通道是否分别在功能上与中间和末端靶受体偶联，从而在募集级联的空间上不同的步骤起作用。(ii)TRPC 6依赖机制的坚定的粘附将详细研究与原子力显微镜技术。最后，我们想调查，是否（iii）TRPC 6通道，也是渗透Na+离子，连接到CXCR 2受体通过修改变构结合的Na+离子的受体蛋白，通过其影响细胞内Na+浓度和/或细胞膜电位。

The recruitment of neutrophil granulocytes from the blood follows a well defined sequence of events including rolling, adhesion and transmigration which is then followed by chemotaxis towards a lesion. Firm adhesion to endothelial cells is integrin-dependent and occurs as a result of activation of G-protein coupled receptors (GPCRs) for chemoattractants (e.g. CXCR2). CXCR2 contains like most other GPCRs an allosteric binding site for Na+ ions. Na+ binding inhibits constitutive GPCR activity which may be as high as 50 % of agonist-stimulated activity. Thus, Na+ binding within GPCRs appears to be a way to ensure the full dynamic range of receptor activation by agonists. CXCR2 ligands, classified as "intermediary chemoattractants", drive chemotaxis of neutrophils towards the vicinity of a lesion. "End target" chemoattractants, released within the lesion and prototypically represented by fMLP, then take over. Thus, chemoattractants act in a spatially distinct order during neutrophil recruitment. Neutrophil recruitment is Ca2+ dependent. We have shown that TRPC6 channels are functionally coupled to CXCR2-dependent steps. However, the contribution of other molecularly defined Ca2+ influx channels to individual steps of the recruitment cascade and the underlying mechanisms are not well characterized. Here we want to test the hypothesis whether (i) TRPC6 and TRPM2 channels are functionally coupled to intermediary and end target receptors, respectively, and thereby act at spatially distinct steps of the recruitment cascade. (ii) TRPC6-dependent mechanisms of firm adhesion will be studied in detail with atomic force microscopic techniques. Finally, we want to investigate, whether (iii) TRPC6 channels which are also permeable to Na+ ions, are linked to CXCR2 receptors by modifying allosteric binding of Na+ ions to the receptor protein via their impact on the intracellular Na+ concentration and/or the cell membrane potential.