Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel.
1. The rectification property of the inward rectifier K+ channel is chiefly due to the block of outward current by cytoplasmic Mg2+ and polyamines. In the cloned inward rectifier K+ channel Kir2.1 (IRK1), Asp172 in the second transmembrane region (M2) and Glu224 in the putative cytoplasmic region after M2 are reported to be critical for the sensitivity to these blockers. However, the difference in the inward rectification properties between Kir2.1 and a very weak inward rectifier sWIRK could not be explained by differences at these two sites. 2. Following sequence comparison of Kir2.1 and sWIRK, we focused this study on Glu299 located in the centre of the putative cytoplasmic region after M2. Single-point mutants of Kir2.1 (Glu224Gly and Glu299Ser) and a double-point mutant (Glu224Gly-Glu299Ser) were made and expressed in Xenopus oocytes or in HEK293T cells. 3. Their electrophysiological properties were compared with those of wild-type (WT) Kir2.1 and the following observations were made. (a) Glu299Ser showed a weaker inward rectification, a slower activation upon hyperpolarization, a slower decay of the outward current upon depolarization, a lower sensitivity to block by cytoplasmic spermine and a smaller single-channel conductance than WT. (b) The features of Glu224Gly were similar to those of Glu299Ser. (c) In the double mutant (Glu224Gly-Glu299Ser), the differences from WT described above were more prominent. 4. These results demonstrate that Glu299 as well as Glu224 control rectification and permeation, and suggest the possibility that the two sites contribute to the inner vestibule of the channel pore. The slowing down of the on- and off-blocking processes by mutation of these sites implies that Glu224 and Glu299 function to facilitate the entry (and exit) of spermine to (and from) the blocking site.
Department of Neurophysiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan. ykubo.phy2@med.tmd.ac.jp
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Kubo
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Murata
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004553:Electric Conductivity; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation / Q000502:physiology; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000235:genetics* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D012474:Salmon; D013096:Spermine / Q000494:pharmacology; D014981:Xenopus
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D013096:Spermine; D008274:Magnesium