四乙铵(TEA),季铵化合物,是一种众所周知的钾离子通道阻滞剂,属于各种亚科,如KV1-3,KCa1,2和原核KcsA。在许多情况下,TEA通过开放孔阻断从细胞外一侧起作用。TEA还可以阻断瞬时受体电位(TRP)阳离子通道,例如TRPM7,以电压依赖的方式。在人类T淋巴细胞中,细胞内(胞质)TEA及其类似物TMA(四甲基铵)在向外而不是向内方向抑制TRPM7通道电流。相比之下,胞内Mg2+,质子和多胺同样抑制外向和内向电流分量。同样,大多数可用的药理学工具以不依赖电压的方式抑制TRPM7通道.由于TRPM7是一个陡峭的向外整流电导,电压依赖性阻断剂可用于研究该通道的细胞功能。TRPM7蛋白在多种细胞系中内源性表达,包括HEK,HeLa,CHO,RBL和Jurkat.使用膜片钳电生理学,我们发现在20mMTEA-Cl存在下孵育HEK293和JurkatT细胞过夜,导致全细胞TRPM7外向电流几乎完全阻断,在闯入时测量。相比之下,在负载TEA的细胞中,内向电流没有变化。在整个细胞中清除细胞内溶液后,阻滞是完全可逆的,而在穿孔的贴片记录配置中却不是。用20mMTMA-Cl孵育过夜导致对向外TRPM7电流的更适度的阻断。内部129mMTMA和TEA消除了大部分向外的电流。转染的HEK293细胞中的TEA摄取导致重组鼠TRPM7和Mg2+和pH不敏感的Ser1107Arg变体的阻断。出乎意料的是,Tris-HCl,广泛使用的pH缓冲液,同样可以加载到Jurkat和HEK细胞中,并优先阻断向外的TRPM7电流。内部溶液中的20mM和129mMTris阻断了向外而不是向内方向的TRPM7电流。TEA的电压依赖性通道阻塞,TMA和Tris负载将用于研究完整细胞中TRPM7介导的离子转运的性质和功能。
Tetraethylammonium (TEA), a quaternary ammonium compound, is a well-known blocker of potassium channels belonging to various subfamilies, such as KV1-3, KCa1, 2 and prokaryotic KcsA. In many cases, TEA acts from the extracellular side by open pore blockade. TEA can also block transient receptor potential (TRP) cation channels, such as TRPM7, in a voltage-dependent manner. In human T lymphocytes, intracellular (cytosolic) TEA and its analog TMA (tetramethylammonium) inhibit TRPM7 channel currents in the outward but not inward direction. By contrast, intracellular Mg2+, protons and polyamines inhibit both outward and inward current components equally. Likewise, the majority of available pharmacological tools inhibit TRPM7 channels in a voltage-independent manner. Since TRPM7 is a steeply outwardly rectifying conductance, voltage-dependent blockers can be useful for studying the cellular functions of this channel. TRPM7 protein is endogenously expressed in diverse cell lines, including HEK, HeLa, CHO, RBL and Jurkat. Using patch-clamp electrophysiology, we found that incubating HEK293 and Jurkat T cells overnight in the presence of 20 mM TEA-Cl, resulted in the nearly complete blockade of whole-cell TRPM7 outward current, measured at break-in. By contrast, the inward current was unchanged in TEA-loaded cells. The blockade was fully reversible after washout of intracellular solution in whole-cell but not in perforated-patch recording configurations. Overnight incubation with 20 mM TMA-Cl resulted in a more modest blockade of the outward TRPM7 current. Internal 129 mM TMA and TEA eliminated most of the outward current. TEA uptake in transfected HEK293 cells led to blockade of recombinant murine TRPM7 and the Mg2+ and pH insensitive Ser1107Arg variant. Unexpectedly, Tris-HCl, a widely used pH buffer, could similarly be loaded into Jurkat and HEK cells, and preferentially blocked outward TRPM7 currents. 20 mM and 129 mM Tris in the internal solution blocked TRPM7 current in outward but not inward direction. Voltage-dependent channel blockade by TEA, TMA and Tris loading will be useful for studying the properties and functions of TRPM7-mediated ion transport in intact cells.