Single-Channel Recordings of K+ Channels Expressed in Saccharomyces Cerevisiae

The study of ion channels has been traditionally based on expression in mammalian cell lines or Xenopus laevis oocytes and their functions have been investigated by various electrophysiological techniques. Recently the structures of several ion channels have been determined and this has increased the understanding of ion channel function. Potassium channels are the largest and most diverse group of ion channels; yet the number of high resolution structures of K+ channels is limited, due to difficulties in their recombinant overexpression and purification. Our group has developed a cost-effective method for stable expression of K+ channels in Saccharomyces cerevisiae, as well as a technique for purification of correctly folded protein. The aim is to explore the functionality of a number of K+ channels produced in yeast, including hERG, Slick, Slack as well as the uncharacterized malaria potassium channels PfKch1 and PfKch2, by the planar patch clamp technique. Channels were tagged with GFP and histidine to follow their expression in Saccharomyces cerevisiae and allow purification by Ni-affinity chromatography, after solubilization in FOS-12. Purified proteins were reconstituted into giant unilamellar vesicles (diphyntanoylphosphatidilcholine + 10% cholesterol) and proteoliposomes were used for recording of single-channels on planar lipid bilayers with a Port-a-Patch (Nanion Technologies). Continued recordings of hERG single-channel currents revealed voltage sensitivity and a slope conductance of 16 pS. Slick and Slack single-channel activity was enhanced by Na+ and showed a conductance of 138 and 140 pS respectively. We measured for the first time full length PfKch2 and a C-terminus truncated PfKch1, which showed Ca2+ modulation and conductance of around 28 and 13 pS, respectively. In conclusion, our high-yield expression of K+ channels expressed in and purified from yeast showed similar current characteristics as single channels previously recorded from mammalian cells, Xenopus oocytes or artificial bilayers.