Villarroel A, Burnashev N, Sakmann B
Max-Planck-Institut für medizinische Forschung, Abteilung Zellphysiologie, Heidelberg, Germany.
Biophys J. 1995 Mar;68(3):866-75. doi: 10.1016/S0006-3495(95)80263-8.
Glutamate-activated single-channel and ensemble currents were recorded from Xenopus laevis oocytes and HEK 293 cells expressing a recombinant NMDA receptor, assembled from NR1 and NR2A subunits. Cesium was the main charge carrier, and organic cations were used to determine the presence of vestibules of this channel and to estimate its pore diameter. The large organic cations tris-(hydroxymethyl)-aminomethane (Tris), N-methyl-glucamine (NMG), arginine (NMG), arginine (Arg), choline, and tetramethylammonium (TMA), when added in millimolar concentrations to the extracellular or cytoplasmic side, produced a voltage-dependent blockade of single-channel Cs+ currents. These molecules behaved as impermeant ions that only partially traverse the channel from either side. The smaller cations trimethylammonium (TriMA) and dimethylammonium (DMA) produced a small and nearly voltage-independent reduction in current amplitude, suggesting that they are permeant. In biionic experiments with Cs+ as the reference ion, the large blocking cations NMG, Arg, Tris, TMA, choline, hexamethonium (Hme), triethylammonium (TriEA), and tetraethylammonium (TEA) showed no measurable permeability. TriMA and smaller ammonium derivatives were permeant. Both the permeability and single-channel conductance of organic cations, relative to Cs+, decreased as the ion size increased. The results suggest that the NMDA receptor has extracellular and cytoplasmic mouths that can accommodate large cations up to 7.3 A in mean diameter. The narrow portion of the pore is estimated to have a mean diameter of 5.5 A.
从表达由NR1和NR2A亚基组装而成的重组N-甲基-D-天冬氨酸受体的非洲爪蟾卵母细胞和人胚肾293细胞中记录了谷氨酸激活的单通道电流和整体电流。铯是主要的电荷载体，使用有机阳离子来确定该通道前庭的存在并估计其孔径。当以毫摩尔浓度添加到细胞外或细胞质侧时，大的有机阳离子三（羟甲基）氨基甲烷（Tris）、N-甲基葡糖胺（NMG）、精氨酸（Arg）、胆碱和四甲基铵（TMA）会产生电压依赖性的单通道Cs⁺电流阻断。这些分子表现为非渗透性离子，仅从任一侧部分穿过通道。较小的阳离子三甲铵（TriMA）和二甲铵（DMA）会使电流幅度产生小的且几乎与电压无关的降低，这表明它们是可渗透的。在以Cs⁺作为参考离子的双离子实验中，大的阻断阳离子NMG、Arg、Tris、TMA、胆碱、六甲铵（Hme）、三乙铵（TriEA）和四乙铵（TEA）没有显示出可测量的渗透性。TriMA和较小的铵衍生物是可渗透的。相对于Cs⁺，有机阳离子的渗透性和单通道电导都随着离子尺寸的增加而降低。结果表明，N-甲基-D-天冬氨酸受体具有细胞外口和细胞质口，平均直径可达7.3埃的大阳离子可以容纳在其中。孔的狭窄部分估计平均直径为5.5埃。
