Taglialatela M, Ficker E, Wible B A, Brown A M
Rammelkamp Center for Research, Case Western University, Cleveland, OH 44109-1998, USA.
EMBO J. 1995 Nov 15;14(22):5532-41. doi: 10.1002/j.1460-2075.1995.tb00240.x.
Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.
内向整流钾通道中离子传导的关键位点直到现在才被发现。IRK1的C末端区域在Mg2+ i阻断和单通道钾离子电导中起关键作用。假定的第二个跨膜结构域（位置172）中的一个带负电荷的天冬氨酸对于细胞质多胺精胺和亚精胺的时间依赖性阻断至关重要。我们现在已将IRK1中C末端的作用定位到一个单一的带负电荷残基（E224）。将E224突变为G、Q和S会大幅降低整流作用。此外，IRK1 E224G突变降低了Mg2+ i和亚精胺的阻断作用，并且与E224Q突变一样，导致明显的单通道钾离子电导显著降低。双突变IRK1 D172N + E224G对亚精胺阻断明显不敏感，显示出与ROMK1相似的亲和力。这些结果与一个模型相符，即位置224处带负电荷的残基E224是IRK1中孔特性的主要决定因素。通过与位置172处带负电荷的残基D172的特异性相互作用，E224有助于形成Mg2+和多胺的结合口袋，这是强内向整流器的一个特征。
