S. cerevisiae strains CY162 (MATα trk1Δ trk2Δ::HIS3 ura3-52 his4-15, his3Δ200) , PAP7111 (CY162 carrying a PMA1::mcherry fusion) was used for the complementation assay and PAP1500 (α ura3-52 trp::GAL10-GAL4 lys2-801 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL)  was used as host for production of PfKch11−1094-GFP and GFP-PfKch2fl channels for purification.
Recombinant plasmid construction
Full-length or fragments of PfKch1 or PfKch2 codon optimized for expression in Xenopus oocytes or Saccharomyces cerevisiae were purchased from Geneart, DE, and GenScript, USA, respectively. All yeast expression plasmids were generated by in vivo homologous recombination in S. cerevisiae between BamHI, HindIII digested pEMBLyex4  and PCR fragments encoding full length or parts of PfKch1 or PfKch2 cDNA. In-frame C- or N-terminal tagging of PfKch11−1094, PfKch1fl or PfKch2fl with yEGFPs  were constructed by in vivo recombination between BamHI, HindIII digested pEMBLyex4 expression vector and PfKch1 or PfKch2 PCR fragments and a GFP PCR fragment amplified with primers adding either N-terminal or C-terminal TEV sites and HIS8 tags. Homologous recombination was achieved by transforming CY162  or PAP1500  according to the method of Gietz and Schiestl . Correct sequences of tagged constructs were verified by sequencing services offered at Eurofins Genomics, Germany, on purified plasmids.
Functional complementation in liquid media
Transformed CY162 yeast cells were grown at 25 °C in liquid synthetic minimal medium containing 2% glucose as sole carbon source and 100 mM KCl. Exponentially growing cells were harvested, washed four times in sterile 18 mΩ H2O and subsequently re-suspended in 18 mΩ H2O to an OD450 = 0.5 and used to inoculate the minimal media containing wells in the growth assay to an OD450 = 0.05. Complementation assays were performed in 96-well plates at a volume of 200 ml. All growth experiments were performed in amino acid supplemented minimal medium containing 2% glucose, 2% galactose and KCl concentrations from 0 mM til 100 mM. The medium was buffered with 100 mM TES/Tris, pH = 6. Plates were incubated at room temperature and OD450 was measured at least twice a day in a plate-reader (Multiskan RC, Thermo Labsystems).
Cultures were set up by inoculating 5 ml of leucine supplemented synthetic minimal (SD) medium and incubated O/N at RT. When saturated, 100 μl were transferred to media with no leucine, to select for high plasmid copy numbers. These cultures were subsequently transferred to 1 l Expression media (SD medium with valine (150 µg/L), tyrosine (30 mg/L), tryptophan (20 mg/L), threonine (200 mg/L), serine (375 mg/L), proline (20 mg/L), phenylalanine (50 mg/L), lysine (30 mg/L), histidine (20 mg/L), glutamic acid (100 mg/L), cysteine (20 mg/L), aspartic acid (100 mg/L), arginine (20 mg/L), alanine (20 mg/L), glycerol (3% v/v) and glucose (0.5% w/v)) to an OD450 of 0.05. Cultures were incubated at room temperature. At OD450 = 1, each culture was separated in two that were temperature equilibrated at 15 °C or 30 °C, and then induced to express the recombinant channels by addition of induction medium (expression medium with 20% galactose instead of 0.5% glucose) at a final concentration of 2% galactose. Cultures were harvested after 12, 24, 48, 72 and 96 h of incubation.
Bioimaging of live cells
The localization of GFP tagged channels in yeast was determined by bioimaging of live cells that had been induced for channel production for 24–48 h. Images were taken using an Optronics Magnafire model S99802 camera coupled to a Nikon Eclipse E600 microscope at a 1000x magnification.
Preparation of crude membrane fractions
Crude yeast membranes were prepared by homogenizing cells with glass beads as described previously . In short, the resuspension of the 1 l cell culture pellets were carried out in 10 ml ice cold lysis buffer with a pH of 7.5 (10% glycerol (v/v), 1 mM EGTA, 1 mM EDTA and 25 mM imidazole) with protease inhibitors (Aprotinin (1 μg/ml), Benzamidine (1 mM), Chymostatin (1 μg/ml), Leupeptin (1 μg/ml), Pepstatin (1 μg/ml) and PMSF (1 mM)). Cell suspensions were vortexed 5 x 1 min and kept on ice between vortexing cycles. The supernatants were collected and the remaining beads washed with lysis buffer several times, to generate samples of 50 ml total volume. A 10 min centrifugation at 3000 rpm and 4 °C, were carried out in an SS-34 Sorvall rotor, to pellet cell debris in the samples. The crude membrane fractions were then isolated by a 1.5 h ultra-centrifugation at 40,000 rpm and 4 °C using a Sorvall 70TI rotor. The pellets containing the crude membranes were then re-suspended in 3 ml lysis buffer containing protease inhibitors (as described above) and homogenized manually with a Potter–Elvehjem homogenizer and kept at −80 °C.
Protein and GFP quantification
A standard BCA assay  was used to determine the total membrane protein content of crude yeast membrane fractions. A kit (Sigma, USA) was used according to the manufacturer´s specifications, applying known concentrations of chicken ovalbumin for the standard curve. The fluorescence content was quantified by resuspending 25 μg crude membrane proteins in a total of 200 μl buffer (10 mM imidazole, 10% glycerol, 200 mM NaCl and 20 mM phosphate) at pH 7.0 in 96 well white microplates (Nucleon Nunc) and measuring their GFP fluorescence. Measurements were done in a Fluoroskan Ascent spectro fluorometer (Thermo Scientific), using 485 nm excitation and 520 nm emision filters. From a previously generated standard curve of purified GFP mixed with yeast membranes [25, 26] fluorescence could be converted to pmol K+ channel-GFP.
Solubilization and detergent screen
Solubilization of crude membranes, from cells grown at 15 °C was done by incubation in buffer A (10% glycerol, 0.5 M NaCl, 10 mM imidazole, 25 mM Tris–HCl, 0.5 mM EDTA and 0.5 mM EGTA pH 7.6) supplemented with protease inhibitors (Aprotinin (1 μg/ml), Benzamidine (1 mM), Chymostatin (1 μg/ml), Leupeptin (1 μg/ml), Pepstatin (1 μg/ml)and PMSF (1 mM)) at protein:detergent:CHS ratios (w/w) of 1:2:0.7; 1:3:1 or 1:4:1.4. The screen included detergents LDAO, Lauryldimethylamine Noxide; FOS-12, n-dodecylphosphocholine; DDM, n-dodecyl-β-d-maltopyranoside;Cymal-5, 5-Cyclohexyl-1-pentyl-β-d-maltoside; C12E8, Octaethylene glycol monododecyl ether; DM, n-decyl-β-d-maltopyranoside; CHAPS, 3-[(3chol-amidopropyl)- dimethylammonio]-1-propane sulfonate/N,N-dimethyl-3-sulfo-N-[3-[[3a,5b,7a,12a)-3,7,12- tri–hydroxy-24-oxocholan-24- yl]amino] propyl]-1-propana-miniumhy-droxide and Octyl glucoside. Detergents were from either Affymetrix, UK and of Anagrade quality or from GLYCON Biochemicals, Germany. Solubilization was carried out under slow rotation at 4 °C for 1 h. Solubilized proteins were collected after pelleting cell debris by 30 min of ultra-centrifugation 70,000 rpm and 4 °C using a Beckman Optima™TLX ultracentrifuge fitted with an S.N. 96U 826 rotor. Fluorescence estimation was done as before in 96 well white microplates, using the spectro fluorometer (Fluoroskan Ascent, Thermo Scientific) with buffer as a blank. Excitation was at 485 nm and emission at 520 nm. The percentage of fluorescence of solubilized sample supernatant, as compared to fluorescence in the initial crude membrane fractions, was taken as a measure of solubilization efficiency.
Fluorescence detection size exclusion chromatography (FSEC) analysis of solubilized crude membranes was done on an ÄKTA Purifier (GE Healthcare, USA) using a Superose 6 10/300 column and FSEC buffer (20 mM TRIS–HCl, 0.15 M NaCl, 0.03% DDM pH 7.5). The samples were analyzed with and without addition of CHS 0.026% (w/v) and/or 5 mM KCl. The Superose 6 10/300 column effluent was coupled to a fluorescence detector (Shimadzu Prominence RF-20A). This facilitated fluorescence measurements and visualization of the GFP protein elution profiles. Molecular weight estimation of the solubilized channels was done by a comparison to the HMW calibration kit from GE Healthcare dissolved in FSEC buffer to a concentration of 20 mg/ml. The molecular masses of the kit components were: Blue Dextran 2000 kDa–the elution volume of which was used as definition of void volume; Thyroglobulin 669 kDA; Ferritin 440 kDa; Aldolase 158 kDa; Conalbumin 75 kDa; Ovalbumin 43 kDa.
Ni–NTA affinity purification
To purify the PfKch11−1094-GFP channel, they were first solubilized in buffer A at a protein:FOS-12:CHS ratio of 1:3:1 (w/w/w) under slow rotation for 2 h at 4 °C and subsequently centrifuged in a Beckman Optima™TL200 ultracentrifuge for 30 min at 70,000 rpm and 4 °C, to get rid of any unsolubilized material. Supernatants containing solubilized membrane proteins were collected and diluted in Buffer A with protease inhibitors, but without EDTA or EGTA, to reach a detergent concentration of 0.75 mg/ml corresponding to 1.5 times CMC for FOS-12 and a CHS concentration of 0.26 mg/ml. The diluted samples were setup to bind to a 5 ml HisTrap™HP (GE healthcare Life science) column, by running it over the column in a loop O/N at a pace of 1 ml/min at 4 °C. The samples were then separated by a 10-500 mM linear imidazole gradient on the ÄKTA Purifier (GE Healthcare, USA) by a 1 ml/min flow. A total of 95 1 ml fractions were collected, and fluorescence in each fraction was determined using a spectro fluorometer (Fluoroskan Ascent, Thermo Scientific, USA) with buffer used to estimate background fluorescence. Excitation was at 485 nm and emission at 520 nm. The GFP-PfKch2 channels were solubilized at a protein:FOS-12:CHS ratio of 1:1:0.3. The procedure was the same as for PfKch11−1094-GFP channels but using a 1 ml HisTrap™HP (GE healthcare Life science) column instead and washing and eluting at a pace of 0.4 ml/min increase in imidazole concentration from 10 mM to 500 mM. A total of 21 fractions of 1 ml each was collected and fluorescence measured as described above. Purified fluorescence peak fractions of PfKch11−1094-GFP and GFP-PfKch2 were subsequently visualized after SDS-PAGE separation in a 4–20% gradient Novex gel (Invitrogen) by Coomassie staining.
Lipid bilayers and single channel recordings
Purified PfKch11−1094-GFP and GFP-PfKch2fl proteins were reconstituted into Giant Unilamellar Vesicles (GUVs) to assess their functional properties essentially as described earlier . GUVs were prepared by an electro formation method using Vesicle Prep Pro (Nanion Technologies, Germany) with 10 mM 1,2-diphyntanoyl-sn-glycero-3-phosphatidylcholine (DPhPC) (Avanti Polar Lipids, USA) and 1 mM cholesterol (Sigma), both of them dissolved in chloroform and using 1 M D-Sorbitol as intra-GUV solution. The average size of GUVs formed by this method ranged between 30 and 100 mm. To prepare proteoliposomes, purified GFP-PfKch2fl protein with a concentration of 33 ng/ml or PfKch11−1094-GFP with a concentration of 17 ng/ml was mixed with the GUV solution and incubated at room temperature for 20 min. Detergents were removed by addition of absorbent Bio-Beads™SM-2 (Bio-Rad) for 1 h at room temperature, followed by an overnight incubation at 4 °C. Bio-Beads™ were discarded after a short spin. The proteoliposome solution was either used right away or stored at 4 °C for up to 4 days prior to recordings. The formation of lipid bilayers, as well as the channel activity was monitored on a Port-a-Patch (Nanion Technologies, Germany). Lipid bilayers were formed on NPC-1 borosilicate glass chips with a resistance of 2–5 MΩ using symmetrical K+ concentrations on both sides of the bilayer (130 mM KCl, 10 mM HEPES, pH = 7). During experiments, Ca2+ was directly added to the upward facing side of the chip to a final concentration of 10 μM. Data were recorded at room temperature (22 °C) at a sampling rate of 50 kHz and filtered using a low-pass Bessel filter at 2 kHz with an EPC-9 HEKA amplifier (HEKA Elektronik, Germany). Single channel events were detected and analyzed in Clampfit 10 (Molecular Devices, USA).