Background/Aims: The replacement of the amino acid valine at position 388 (Shaker position 438) in hK(v)1.3 channels or at the homologue position 370 in hK(v)1.2 channels resulted in a channel with two different ion conducting pathways: One pathway was the central, potassium-selective alpha-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hK(v)1.3_V388C channel and CTX or MTX in the hK(v)1.2_V370C channel). The other pathway (sigma-pore) was behind the central alpha-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central alpha-pore was closed. In addition, current through the sigma-pore could not be reduced by CTX, KTX or MTX in the hK(v)1.3_V388C or the hK(v)1.2_V370C channel, respectively. Methods: For a more detailed characterization of the sigma-pore, we created a trimer consisting of three hK(v)1.3_V388C alpha-subunits linked together and characterized current through this trimeric hK(v)1.3_V388C channel. Additionally, we determined which amino acids line the sigma-pore in the tetrameric hK(v)1.3_V388C channel by replacing single amino acids in the tetrameric hK(v)1.3_V388C mutant channel that could be involved in sigma-pore formation. Results: Overexpression of the trimeric hK(v)1.3_V388C channel in COS-7 cells yielded typical sigma-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hK(v)1.3_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 sigma-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 hK(v)1.3_V388C background channel. Two of these tetrameric double mutant channels (hK(v)1.3_V388C_T392Y and hK(v)1.3_V388C_Y395W) did not show currents through the sigma-pore. Conclusions: From our experiments with the trimeric hK(v)1.3_V388C channel we conclude that the sigma-pore exists in hK(v)1.3_V388C channels independently of the alpha-pore. From our site-directed mutagenesis experiments in the tetrameric hK(v)1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the sigma-pore. (C) 2018 The Author(s) Published by S. Karger AG, Basel
