マウス内向き整流Kチャネル(Kir2.1)細胞外ループのアスパラギン酸中性化の影響 Effects of neutralization of aspartate residues in the extracellular loops of the murine inwardly rectifying K<SUP>+</SUP> channel, Kir2.1

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Abstract

Removal of external K<SUP>+</SUP> ions abolishes not only inward currents but also outward currents through the inwardly rectifying K<SUP>+</SUP> channel which determines the resting potential. This suggests that external K<SUP>+</SUP> ions are essential for the channel activation. To investigate how extracellular K<SUP>+</SUP> ions act the channel, we made single points mutants in which one of negative charged amino acids in the extracellular loops of the murine inwardly rectifying K<SUP>+</SUP> channel (Kir2.1) was neutralized. cDNA was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. COS-1 cells transfected with D112N did not show inward rectification under whole-cell recording in the normal Tyrode solution. Cells transfected with tandem tetramers with one wild-type (WT) and three D112N mutant subunits (WT-(D112N)3) also did show inward rectification, while cells transfected with tandem tetramers with two wild-type and two D112N subunits (WT2-(D112N)2) showed inward rectification. Channels from WT2-(D112N)2 had the single-channel conductance similar to that of wild-type channels. It is suggested that two negative charges at the D112 site may be required for K<SUP>+</SUP> ions to bind to and activate the channel. <b>[Jpn J Physiol 54 Suppl:S133 (2004)]</b>

Removal of external K<SUP>+</SUP> ions abolishes not only inward currents but also outward currents through the inwardly rectifying K<SUP>+</SUP> channel which determines the resting potential. This suggests that external K<SUP>+</SUP> ions are essential for the channel activation. To investigate how extracellular K<SUP>+</SUP> ions act the channel, we made single points mutants in which one of negative charged amino acids in the extracellular loops of the murine inwardly rectifying K<SUP>+</SUP> channel (Kir2.1) was neutralized. cDNA was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. COS-1 cells transfected with D112N did not show inward rectification under whole-cell recording in the normal Tyrode solution. Cells transfected with tandem tetramers with one wild-type (WT) and three D112N mutant subunits (WT-(D112N)3) also did show inward rectification, while cells transfected with tandem tetramers with two wild-type and two D112N subunits (WT2-(D112N)2) showed inward rectification. Channels from WT2-(D112N)2 had the single-channel conductance similar to that of wild-type channels. It is suggested that two negative charges at the D112 site may be required for K<SUP>+</SUP> ions to bind to and activate the channel. <b>[Jpn J Physiol 54 Suppl:S133 (2004)]</b>

Journal

  • Proceedings of Annual Meeting of the Physiological Society of Japan

    Proceedings of Annual Meeting of the Physiological Society of Japan 2004(0), S133-S133, 2004

    PHYSIOLOGICAL SOCIETY OF JAPAN

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