McNicholas C M, Yang Y, Giebisch G, Hebert S C
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026, USA.
Am J Physiol. 1996 Aug;271(2 Pt 2):F275-85. doi: 10.1152/ajprenal.1996.271.2.F275.
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 (IKATP). 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 IKATP 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.
ATP敏感性内向整流钾通道存在于远端肾单位的顶端膜中，在钾离子循环和分泌中起主要作用。克隆的肾钾通道ROMK1是肾上皮钾通道的一个候选者，因为它与天然通道具有许多功能特性。此外，ROMK1含有一个假定的羧基末端ATP结合位点。尽管在通道活性降低后，胞质Mg-ATP可使ROMK1通道活性重新激活，但核苷酸在通道门控中的作用尚不太明确。我们现在发现，ROMK通道基因的一个选择性剪接转录本ROMK2编码一个氨基末端截短的钾通道，它表达一种ATP调节且对ATP敏感的钾通道（IKATP）。ROMK亚型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IKATP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环）内特定残基的点突变。这些突变要么增强要么减弱了对Mg-ATP激活和抑制的敏感性，从而证明了核苷酸与形成通道的多肽之间存在直接相互作用。
