Manning Declan, Barrett-Jolley Richard, Evans Richard L, Dart Caroline
Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, United Kingdom.
Front Physiol. 2023 Mar 6;14:1141006. doi: 10.3389/fphys.2023.1141006. eCollection 2023.
Skin is the largest organ in the human body with ∼95% of its surface made up of keratinocytes. These cells maintain a healthy skin barrier through regulated differentiation driven by Ca-transcriptional coupling. Many important skin conditions arise from disruption of this process although not all stages are fully understood. We know that elevated extracellular Ca at the skin surface is detected by keratinocyte Gα-coupled receptors that signal to empty endoplasmic reticulum Ca stores. Orai channel store-operated Ca entry (SOCE) and Ca influx "canonical" transient receptor potential (TRPC)-composed channels then activates transcription factors that drive differentiation. While STIM-mediated activation of Orai channels following store depletion is well defined, how TRPC channels are activated is less clear. Multiple modes of TRPC channel activation have been proposed, including 1) independent TRPC activation by STIM, 2) formation of Orai-TRPC-STIM complexes, and 3) the insertion of constitutively-active TRPC channels into the membrane during SOCE. To help distinguish between these models, we used high-resolution microscopy of intact keratinocyte (HaCaT) cells and immunogold transmission electron microscopy (TEM) of HaCaT plasma membrane sheets. Our data shows no evidence of significant insertion of Orai1 or TRPC subunits into the membrane during SOCE. Analysis of transmission electron microscopy data shows that during store-depletion and SOCE, Orai1 and TRPC subunits form separate membrane-localized clusters that migrate towards each other. This clustering of TRPC channel subunits in keratinocytes may support the formation of TRPC-STIM interactions at ER-plasma membrane junctions that are distinct from Orai-STIM junctions.
皮肤是人体最大的器官，其表面约95%由角质形成细胞组成。这些细胞通过由钙转录偶联驱动的调节分化来维持健康的皮肤屏障。许多重要的皮肤疾病都源于这一过程的破坏，尽管并非所有阶段都完全清楚。我们知道，角质形成细胞的Gα偶联受体可检测到皮肤表面细胞外钙升高，该受体发出信号使内质网钙库排空。然后，Orai通道储存式钙内流（SOCE）和由钙内流“典型”瞬时受体电位（TRPC）组成的通道激活驱动分化的转录因子。虽然储存耗竭后STIM介导的Orai通道激活已得到充分明确，但TRPC通道如何被激活尚不清楚。已经提出了多种TRPC通道激活模式，包括1）STIM独立激活TRPC，2）形成Orai-TRPC-STIM复合物，以及3）在SOCE期间将组成型活性TRPC通道插入膜中。为了帮助区分这些模型，我们使用了完整角质形成细胞（HaCaT）的高分辨率显微镜和HaCaT质膜片的免疫金透射电子显微镜（TEM）。我们的数据显示，在SOCE期间没有证据表明Orai1或TRPC亚基大量插入膜中。对透射电子显微镜数据的分析表明，在储存耗竭和SOCE期间，Orai1和TRPC亚基形成单独的膜定位簇，它们相互靠近。角质形成细胞中TRPC通道亚基的这种聚集可能支持在ER-质膜连接处形成与Orai-STIM连接处不同的TRPC-STIM相互作用。
