The Lectin LecA Sensitizes the Human Stretch-Activated Channel TREK-1 but Not Piezo1 and Binds Selectively to Cardiac Non-myocytes
The Lectin LecA Sensitizes the Human Stretch-Activated Channel TREK-1 but Not Piezo1 and Binds Selectively to Cardiac Non-myocytes
复制标题
DOI：
10.3389/fphys.2020.00457
复制
发表时间：
2020-05-15
影响因子：
4
通讯作者：
Peyronnet, Remi
中科院分区：
文献类型：
--
作者：
Darkow, Elisa;Rog-Zielinska, Eva A.;Peyronnet, Remi
健康的心脏不断适应一系列复杂的动态变化的机械条件。机械环境被多种心脏疾病改变并促成多种心脏疾病。机械刺激由细胞机械传感器检测和转导，包括拉伸激活离子通道（SAC）。SAC在心脏中的确切作用尚不清楚，部分原因是SAC特异性药理学调节剂很少。也就是说，大多数SAC可以被膜弯曲的诱导物激活。凝集素LecA是铜绿假单胞菌的一种毒力因子，是铜绿假单胞菌生长所必需的。产气荚膜梭菌引起的膜弯曲，导致内吞结构的形成和细菌细胞侵入。我们调查是否LecA调制SAC活动。选择TREK-1和Piezo 1是因为它们在包括心脏组织在内的体内广泛表达，并且它们分别是钾选择性和阳离子非选择性SAC家族的“典型代表”。活细胞共聚焦显微镜和电子断层成像被用来跟踪LecA的结合动力学，并跟踪细胞形态和膜拓扑结构在人胚肾（HEK）细胞和巨单层囊泡（GUV）的变化。进一步用人TREK-1或Piezo 1构建体转染HEK细胞，并使用膜片钳技术记录离子通道活性。最后，新鲜分离的心肌细胞用于研究LecA结合的细胞类型依赖性。LecA（500 nM）在几秒钟内结合到HEK细胞表面，在细胞-细胞接触部位浓度最高。在LecA存在下，在细胞的质膜（通过LecA暴露17分钟）以及GUV中检测到局部膜内陷。在HEK细胞中，LecA使TREK-1对电压和机械刺激敏感，但不使Piezo 1敏感。在新鲜分离的心肌细胞中，LecA与非心肌细胞结合，但不与心室或心房心肌细胞结合。在小鼠、兔、猪和人的心肌细胞中观察到这种细胞类型特异性缺乏结合。我们的研究结果表明，LecA可以作为一种药理学工具，以细胞类型优先的方式研究SAC。这可能有助于基于组织的研究SAC在心脏非肌细胞中的作用。
The healthy heart adapts continuously to a complex set of dynamically changing mechanical conditions. The mechanical environment is altered by, and contributes to, multiple cardiac diseases. Mechanical stimuli are detected and transduced by cellular mechano-sensors, including stretch-activated ion channels (SAC). The precise role of SAC in the heart is unclear, in part because there are few SAC-specific pharmacological modulators. That said, most SAC can be activated by inducers of membrane curvature. The lectin LecA is a virulence factor ofPseudomonas aeruginosaand essential forP. aeruginosa-induced membrane curvature, resulting in formation of endocytic structures and bacterial cell invasion. We investigate whether LecA modulates SAC activity. TREK-1 and Piezo1 have been selected, as they are widely expressed in the body, including cardiac tissue, and they are "canonical representatives" for the potassium selective and the cation non-selective SAC families, respectively. Live cell confocal microscopy and electron tomographic imaging were used to follow binding dynamics of LecA, and to track changes in cell morphology and membrane topology in human embryonic kidney (HEK) cells and in giant unilamellar vesicles (GUV). HEK cells were further transfected with human TREK-1 or Piezo1 constructs, and ion channel activity was recorded using the patch-clamp technique. Finally, freshly isolated cardiac cells were used for studies into cell type dependency of LecA binding. LecA (500 nM) binds within seconds to the surface of HEK cells, with highest concentration at cell-cell contact sites. Local membrane invaginations are detected in the presence of LecA, both in the plasma membrane of cells (by 17 min of LecA exposure) as well as in GUV. In HEK cells, LecA sensitizes TREK-1, but not Piezo1, to voltage and mechanical stimulation. In freshly isolated cardiac cells, LecA binds to non-myocytes, but not to ventricular or atrial cardiomyocytes. This cell type specific lack of binding is observed across cardiomyocytes from mouse, rabbit, pig, and human. Our results suggest that LecA may serve as a pharmacological tool to study SAC in a cell type-preferential manner. This could aid tissue-based research into the roles of SAC in cardiac non-myocytes.