マウス内向き整流Kチャネル (Kir2.1) 細胞外ループの酸性アミノ酸中性化の影響 Effects of neutralization of negatively charged amino acid 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 point mutants neutralized at negatively charged amino acid residues in the extracellular loops of the murine inwardly rectifying K<SUP>+</SUP> channel (Kir2.1). cDNA was transfected into COS-1 or HEK293 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. Single point mutants (D112N, D114N, E125Q, D152N and E153Q) showed functional expression, and the gating properties and the unitary conductance of the mutants (D112N, D114N and D152N) were similar to those of the wild type (WT). Double point mutation of D152N/E153Q eliminated the ionic conductance of Kir2.1. Channels from tandem tetramers of Kir2.1 with one WT and three D152N/E153Q mutant subunits (WT-(D152N/E153Q)3) showed inward rectification. However, the whole-cell current density of the mutant was significantly smaller than that of wild-type channels in Tyrode solution. It is suggested that two negative charges at the D152 and E153 sites may be required for K<SUP>+</SUP> ions to bind to and activate the channel. <b>[Jpn J Physiol 55 Suppl:S134 (2005)]</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 point mutants neutralized at negatively charged amino acid residues in the extracellular loops of the murine inwardly rectifying K<SUP>+</SUP> channel (Kir2.1). cDNA was transfected into COS-1 or HEK293 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. Single point mutants (D112N, D114N, E125Q, D152N and E153Q) showed functional expression, and the gating properties and the unitary conductance of the mutants (D112N, D114N and D152N) were similar to those of the wild type (WT). Double point mutation of D152N/E153Q eliminated the ionic conductance of Kir2.1. Channels from tandem tetramers of Kir2.1 with one WT and three D152N/E153Q mutant subunits (WT-(D152N/E153Q)3) showed inward rectification. However, the whole-cell current density of the mutant was significantly smaller than that of wild-type channels in Tyrode solution. It is suggested that two negative charges at the D152 and E153 sites may be required for K<SUP>+</SUP> ions to bind to and activate the channel. <b>[Jpn J Physiol 55 Suppl:S134 (2005)]</b>

Journal

  • Proceedings of Annual Meeting of the Physiological Society of Japan

    Proceedings of Annual Meeting of the Physiological Society of Japan 2005(0), S134-S134, 2005

    PHYSIOLOGICAL SOCIETY OF JAPAN

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