The role of TRPC6 channels in neutrophil recruitment

从血液中募集中性粒细胞遵循一系列明确的事件，包括滚动、粘附和迁移，然后向病变趋化。与内皮细胞的牢固粘附依赖于整合素，是g蛋白偶联受体（gpcr）对化学引诱剂（如CXCR2）激活的结果。CXCR2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，其活性可能高达激动剂刺激活性的50%。因此，gpcr内的Na+结合似乎是一种确保受体被激动剂激活的全动态范围的方法。CXCR2配体，被归类为“中间趋化剂”，驱动中性粒细胞向病变附近趋化。“最终目标”化学引诱剂，在病灶内释放，典型地以fMLP为代表，然后接管。因此，在中性粒细胞募集过程中，趋化剂以不同的空间顺序起作用。中性粒细胞的募集依赖于Ca2+。我们已经证明TRPC6通道与cxcr2依赖性步骤在功能上耦合。然而，其他分子定义的Ca2+内流通道对招募级联的各个步骤的贡献及其潜在机制尚未得到很好的表征。在这里，我们想要验证以下假设：(i) TRPC6和TRPM2通道是否分别与中间和最终目标受体功能偶联，从而在招募级联的空间不同步骤中起作用。（ii）依赖trpc6的牢固粘附机制将通过原子力显微技术进行详细研究。最后，我们想要研究（iii） TRPC6通道是否通过Na+离子对细胞内Na+浓度和/或细胞膜电位的影响，通过改变Na+离子与受体蛋白的变构结合而与CXCR2受体相连，TRPC6通道也可渗透到Na+离子中。

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.