A Near-Ir Fluorescent Sensor for Detecting Cellular Potassium Efflux

Abstract

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.