Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells
Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells
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DOI：
10.1371/journal.pone.0009447
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发表时间：
2010-02-26
期刊：
影响因子：
3.7
通讯作者：
Thomas, Serge L. Y.
中科院分区：
文献类型：
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作者：
Dyrda, Agnieszka;Cytlak, Urszula;Thomas, Serge L. Y.
背景：红细胞的力学、流变学和形状特性是由它们的皮质细胞骨架决定的，进化优化的皮质细胞骨架提供了通过比细胞直径窄得多的毛细血管所需的动态变形能力。由这种流动引起的剪切应力，以及在某些病理条件下产生的局部膜变形，如镰状细胞性贫血，已被证明可以增加膜的通透性，这主要是基于红细胞悬浮液的实验。在这里，我们尝试在细胞上膜片钳下对单个红细胞中由局部和受控膜变形激活的膜电流进行首次测量，以确定拉伸激活电流的性质。方法/主要发现：膜片钳技术的细胞附着结构用于记录完整红细胞的单通道活性。在膜变形和不变形的情况下均获得了千兆赫的密封层。施加10毫米汞柱的负压脉冲，持续时间不超过5秒，就会引起变形。只有当膜在移液管内的负压下呈圆顶状时，才能检测到电流。K+和Cl-电流严格依赖于Ca2+的存在。Ca2+依赖电流是短暂的，典型的衰减一半时间约为5-10分钟，表明拉伸激活Ca2+通透性（PCa）的自发失活。这些结果表明，局部膜变形可以瞬间激活Ca2+通透性通路，导致[Ca2+]增加(i)， Ca2+敏感的K+通道（Gardos通道，IK1, KCa3.1）的二次激活，以及超极化诱导的阴离子电流。结论/意义：在局部膜变形下观察到的拉伸激活的短暂性PCa可能是红细胞正常衰老过程中Ca2+介导效应的原因，也是某些遗传性贫血（如地中海贫血和镰状细胞性贫血）中红细胞Ca2+含量增加的原因。
Background: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.Methodology/Principal Findings: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patchpipette. K+ and Cl- currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+](i), secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.Conclusions/Significance: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.