Current through the σ-pore was first detected in hKv1.3_V388C channels, where the V388C mutation in hKv1.3 channels opened a new pathway (σ-pore) behind the central α-pore. Typical for this mutant channel was inward current at potentials more negative than -100 mV when the central α-pore was closed. The α-pore blockers such as TEA+ and peptide toxins (CTX, MTX) could not reduce current through the σ-pore of hKv1.3_V388C channels. This new pathway would proceed in parallel to the α-pore in the S6-S6 interface gap. To see whether this phenomenon is restricted to hKv1.3 channels we mutated hKv1.2 at the homologue position (hKv1.2_V370C). By overexpression of hKv1.2_V370C mutant channels in COS-7 cells we could show typical σ-currents. The electrophysiological properties of the σ-pore in hKv1.3_V388C and hKv1.2_V370C mutant channels were similar. The σ-pore of hKv1.2_V370C channels was most permeable to Na+ and Li+ whereas Cl- and protons did not influence current through the σ-pore. Tetraethylammonium (TEA+), charybdotoxin (CTX) and maurotoxin (MTX), known α-pore blockers, could not reduce current through the σ-pore of hKv1.2_V370C channels. Taken together we conclude that the observation of σ-pore currents is not restricted to Kv1.3 potassium channels but can also be observed in a closely related potassium channel. This finding could have implications in the treatment of different ion channel diseases linked to mutations of the respective channels in regions close to homologue position investigated by us.

Background/Aims: The replacement of the amino acid valine at position 388 (Shaker position 438) in hK_v1.3 channels or at the homologue position 370 in hK_v1.2 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 hK_v1.3_V388C channel and CTX or MTX in the hK_v1.2_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 hK_v1.3_V388C or the hK_v1.2_V370C channel, respectively. Methods: For a more detailed characterization of the σ-pore, we created a trimer consisting of three hK_v1.3_V388C α-subunits linked together and characterized current through this trimeric hK_v1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hK_v1.3_V388C channel by replacing single amino acids in the tetrameric hK_v1.3_V388C mutant channel that could be involved in σ-pore formation. Results: Overexpression of the trimeric hK_v1.3_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 hK_v1.3_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 hK_v1.3_V388C background channel. Two of these tetrameric double mutant channels (hK_v1.3_V388C_T392Y and hK_v1.3_V388C_Y395W) did not show currents through the σ-pore. Conclusions: From our experiments with the trimeric hK_v1.3_V388C channel we conclude that the σ-pore exists in hK_v1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hK_v1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

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The mutant hKv1.3_V388C channel shows inward current at potentials more negative than 100 mV when the central apore is closed (Pruetting et al., JBC 286:20031-20042). We believe that the V388C mutation in hKv1.3 channels opens a new pathway (s-pore) behind the K þ conducting apore. The entry of the spore from the extracellular side is presumably located at the backside of Y395 (Shaker position 445) and F384 (Shaker position 434). One spore seems to be formed between two neighboring S6 subunits and it runs parallel to the apore. For a more detailed characterization of the spore, we created a concatamer consisting of three hKv1.3_V388C asubunits linked together. Overexpression of this trimeric hKv1.3_V388C channel in COS7 cells yielded typical spore 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 channels were similar: currents 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 spore was mostly permeable to Na þ and Li þ. For the determination of the path of the spore in the hKv1.3_V388C channel we mutated single amino acids in the hKv1.3_V388C mutant channel that maybe involved in spore formation. Two of these mutant channels (hKv1.3_V388C_W384F, hKv1.3_V388C_Y395W) reduced current through the spore indicating that these positions might line the spore.