Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels

Cellular Physiology and Biochemistry

Pavel Tyutyaev¹ 

关键词: Potassium channel;    Kv1.3 channel;    σ-pore;    Electrophysiology;    Patch-clamp;   

DOI  :  10.1159/000488840

学科分类：分子生物学，细胞生物学和基因

来源: S Karger AG

【 摘 要 】

Background/Aims The replacement of the amino acid valine at position 388 (Shaker position 438) in hKv1.3 channels or at the homologue position 370 in hKv1.2 channels resulted in a channel with two different ion conducting pathways One pathway was the central, potassium-selective α-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hKv1.3_V388C channel and CTX or MTX in the hKv1.2_V370C channel). The other pathway (σ-pore) was behind the central α-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central α-pore was closed. In addition, current through the σ-pore could not be reduced by CTX, KTX or MTX in the hKv1.3_V388C or the hKv1.2_V370C channel, respectively. Methods For a more detailed characterization of the σ-pore, we created a trimer consisting of three hKv1.3_V388C α-subunits linked together and characterized current through this trimeric hKv1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hKv1.3_V388C channel by replacing single amino acids in the tetrameric hKv1.3_V388C mutant channel that could be involved in σ-pore formation. Results Overexpression of the trimeric hKv1.3_V388C channel in COS-7 cells yielded typical σ-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channel were similar currents could be observed at potentials more negative than -100 mV, were not carried by protons or chloride ions, and could not be reduced by peptide toxins (CTX, MTX) or TEA. The σ-pore was mostly permeable to Na+ and Li+. In addition, in our site-directed mutagenesis experiments, we created a number of new double mutant channels in the tetrameric hKv1.3_V388C background channel. Two of these tetrameric double mutant channels (hKv1.3_V388C_T392Y and hKv1.3_V388C_Y395W) did not show currents through the σ-pore. Conclusions From our experiments with the trimeric hKv1.3_V388C channel we conclude that the σ-pore exists in hKv1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hKv1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

【 授权许可】

CC BY-NC-ND