Pottosin Igor, Delgado-Enciso Iván, Bonales-Alatorre Edgar, Nieto-Pescador María G, Moreno-Galindo Eloy G, Dobrovinskaya Oxana
Center for Biomedical Research, University of Colima, Colima, Mexico.
School of Medicine, University of Colima, Colima, Mexico.
Biochim Biophys Acta. 2015 Jan;1848(1 Pt A):51-9. doi: 10.1016/j.bbamem.2014.09.008. Epub 2014 Sep 28.
Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of 50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa1) and were equally permeable for Ca²⁺ and Mg²⁺. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³⁺ (10 μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 μM) and SKF 96365 (250 μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²⁺ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.
机械敏感通道几乎存在于每一个活细胞中，但在T淋巴细胞中其功能存在的证据却缺失。在本研究中，我们采用膜片钳技术的吸附式和内面向外式模式，对Jurkat T淋巴母细胞中被膜拉伸快速激活的阳离子通道进行了表征。在约50mmHg的负压下实现了半激活。在吸附式模式下，单通道电流呈现内向整流，在零指令电压下的单位电导约为40 pS。在切除的内面向外膜片中，整流转变为外向整流。机械敏感通道对单价和二价阳离子的区分较弱（PCa/PNa~1），对Ca²⁺和Mg²⁺的通透性相同。药理学分析表明，机械敏感通道被氨氯地平（1mM）和Gd³⁺（10 μM）以电压依赖性方式有效阻断。它们也几乎被钌红（1 μM）和SKF 96365（250 μM）完全阻断，这两种物质分别是瞬时受体电位香草酸亚型2（TRPV2）通道的抑制剂。同时，这些通道对2-氨基乙氧基二苯硼酸（2-APB，100 μM）或N-（对戊基肉桂酰基）邻氨基苯甲酸（ACA，50 μM）不敏感，它们分别是瞬时受体电位香草酸亚型1（TRPC）或瞬时受体电位褪黑素亚型（TRPM）通道的拮抗剂。人TRPV2小干扰RNA几乎消除了拉伸激活电流。TRPV2是具有多方面功能和调节机制的通道，在淋巴细胞Ca²⁺信号传导中可能具有重要作用。本文在淋巴细胞功能的背景下讨论了机械应力对其调节的意义。
