Vargas Anibal A, Cisterna Bruno A, Saavedra-Leiva Fujiko, Urrutia Carolina, Cea Luis A, Vielma Alex H, Gutierrez-Maldonado Sebastian E, Martin Alberto J M, Pareja-Barrueto Claudia, Escalona Yerko, Schmachtenberg Oliver, Lagos Carlos F, Perez-Acle Tomas, Sáez Juan C
Departamento de Fisiología, Pontificia Universidad Católica de Chile Santiago, Chile.
Departamento de Fisiología, Pontificia Universidad Católica de ChileSantiago, Chile; Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de ValparaísoValparaíso, Chile.
Front Physiol. 2017 Feb 9;8:38. doi: 10.3389/fphys.2017.00038. eCollection 2017.
Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs under physiological and pathological conditions in cells that express them.
尽管连接蛋白（Cxs）在哺乳动物机体的细胞中广泛表达，但Cx39的表达模式非常有限，基于Cx39的通道[半通道（HCs）和间隙连接通道（GJCs）]的生物物理特性在很大程度上仍不清楚。在此，我们使用转染了Cx39的HeLa细胞（HeLa-Cx39细胞），在其中未检测到细胞间电偶联，这表明不存在GJCs。然而，在暴露于已知可增加其他Cx半通道开放概率的条件下的细胞表面发现了功能性半通道（例如，细胞外无二价阳离子溶液（DCFS）、细胞外碱性pH值、机械刺激以及去极化至正膜电位）。Cx39半通道被一些传统的Cx半通道阻滞剂阻断，但不被其他阻滞剂或泛连接蛋白1通道阻滞剂阻断。HeLa-Cx39细胞显示出与亲代细胞相似的静息膜电位（RMPs），并且暴露于DCFS会降低Cx39转染细胞中的RMPs，但亲代细胞中则不会。在这些条件下，约75 pS的单位事件在HeLa-Cx39细胞中很常见，而在亲代细胞中不存在。对具有不同物理化学特征的染料进行实时细胞摄取实验，以及应用机器学习方法表明，Cx39半通道优先通透具有六类描述符特征化的分子，即：（1）电负性，（2）电离势，（3）极化率，（4）大小和几何形状，（5）拓扑柔韧性和（6）化合价。然而，由机械刺激或碱性pH值打开的Cx39半通道对Ca不通透。基于Cx39的通道的分子模型表明，在对螺旋区域的细胞内部分存在的一个收缩与一个电负性斑块共定位，形成一个能量和空间屏障，在GJCs的情况下可能会阻碍通道功能。此处报道的结果表明Cx39形成半通道，并增进了我们对表达Cx39的细胞在生理和病理条件下半通道功能作用的理解。
