Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2)
Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2)
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DOI：
10.1152/ajprenal.1996.271.2.f275
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发表时间：
1996-08-01
影响因子：
4.2
通讯作者：
Hebert, SC
中科院分区：
文献类型：
--
作者：
McNicholas, CM;Yang, YH;Hebert, SC
atp敏感的、向内矫正的K+通道存在于远端肾元的根尖膜中，在K+的循环和分泌中起主要作用。克隆肾K+通道ROMK1是肾上皮K+通道的候选通道，因为它与天然通道具有许多功能特征。此外，ROMK1含有一个假定的羧基末端atp结合位点。虽然ROMK1通道的活性可以通过细胞质Mg-ATP重新激活，但核苷酸在通道门控中的作用尚不确定。我们现在展示了ROMK通道基因的选择性剪接转录本ROMK2，它编码具有截断氨基末端的K+通道，表达atp调节和atp敏感的K+通道（I-KATP）。ROMK同工异构体氨基末端的差异改变了通道门控机制对ATP的敏感性。为了测试肾脏I-KATP的ATP敏感性是否由核苷酸的直接相互作用介导，我们研究了ROMK2磷酸环（P-loop）内特定残基的点突变。这些增强或减弱了对Mg-ATP激活和抑制的敏感性，从而证明了核苷酸与通道形成多肽的直接相互作用。
ATP-sensitive, inwardly rectifying K+ channels are present in apical membranes of the distal nephron and play a major role in K+ recycling and secretion. The cloned renal K+ channel, ROMK1, is a candidate for the renal epithelial K+ channel, since it shares many functional characteristics with the native channel. Additionally, ROMK1 contains a putative carboxy-terminal ATP-binding site. Although ROMK1 channel activity could be reactivated by cytosolic Mg-ATP after rundown, the role of nucleotides in channel gating was less certain. We now show that an alternatively spliced transcript of the ROMK channel gene, ROMK2, which encodes a K+ channel with a truncated amino terminus, expresses an ATP-regulated and ATP-sensitive K+ channel (I-KATP). Differences in the amino terminus of ROMK isoforms alters the sensitivity of the channel-gating mechanism to ATP. To test whether ATP sensitivity of renal I-KATP is mediated by direct interaction of nucleotide, point mutation of specific residues within the ROMK2 phosphate loop (P-loop) were investigated. These either enhanced or attenuated the sensitivity to both activation and inhibition by Mg-ATP, thus demonstrating a direct interaction of nucleotide with the channel-forming polypeptide.