Mutations in the pore region of ROMK enhance Ba2+ block
Mutations in the pore region of ROMK enhance Ba2+ block
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DOI：
10.1152/ajpcell.1996.271.6.c1949
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发表时间：
1996-12-01
影响因子：
5.5
通讯作者：
Sackin, H
中科院分区：
文献类型：
--
作者：
Zhou, H;Chepilko, S;Sackin, H
通过将两个氨基酸亮氨酸（L）117和缬氨酸（V）121分别改变为异亮氨酸（I）和苏氨酸（T），改变来自肾脏的K+选择性通道（ROMK 2）的疏水性“P "（孔）区域的序列，以匹配密切相关的内向整流（IRK 1）通道。在非洲爪蟾卵母细胞中表达的突变通道在Ba 2+存在下在零电压下具有0.07 +/- 0.01 mM的表观抑制常数[Ki（0）]，其比野生型通道的Ki（0）（4.7 +/- 1.0 mM）低50倍以上。对Ba ~（2+）的敏感性增加是由点突变V121 T引起的。单通道测量表明，增加的亲和力涉及增加的对Ba 2+块的速率和降低的关闭速率。Csi的封锁也得到了加强。L117 I/V121 T突变体的单通道电导增加了50%，而内向整流的程度，离子选择性和对K+的表观亲和力基本不变。当中性天冬酰胺残基内的第二个假定的跨膜结构域的ROMK通道被替换为天冬氨酸，相应的氨基酸在IRK 1，内向整流的程度增强，但Ba 2+块和单通道内向电导不受影响。因此，Ba 2+结合的网站似乎是不同的内部Mg 2+块的轨迹和至少一个网站，确定K+电导率。
The sequence of the hydrophobic ''P'' (pore) region of a K+-selective channel from the kidney (ROMK2) was altered to match that of the closely related inward rectifier (IRK1) channel by changing two amino acids, leucine (L) 117 and valine (V) 121, to isoleucine (I) and threonine (T), respectively. The mutant channel expressed in Xenopus laevis oocytes had an apparent inhibition constant at zero voltage [K-i(0)] in the presence of Ba2+ of 0.07 +/- 0.01 mM, which was more than 50 times lower than the K-i(0) of the wild-type channel (4.7 +/- 1.0 mM). The increased sensitivity to Ba2+ was accounted for by the point mutation V121T. Single-channel measurements indicated that the increased affinity involved an increase in the on-rate for Ba2+ block and a decrease in the off-rate. Block by Csi was also enhanced. The single-channel conductance of the L117I/V121T mutant was increased by 50%, whereas the degree of inward rectification, ion selectivity, and apparent affinity for K+ were essentially unchanged. When the neutral asparagine residue within the second putative membrane-spanning domain of the ROMK channel was substituted with aspartic acid, the corresponding amino acid in IRK1, the degree of inward rectification was enhanced but Ba2+ block and single-channel inward conductance were unaffected. Thus the site of Ba2+ binding appears to be distinct from the locus of internal Mg2+ block and from at least one of the sites that determines K+ conductivity.