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Su et al. 10.1073/pnas.1602815113 SI Methods then a thrombin protease recognition sequence to the N terminus Constructs. DNA fragments encoding different constructs were of cloned genes. amplified by PCR using Phusion DNA polymerase (New England hSlo1 was cloned into RML6 vector and was expressed in Biolabs) and cloned into expression vectors modified by the CHO cells. MacKinnon laboratory for yeast, insect, and mammalian cells. All mTRAAK chimeric construct used in protein purification was constructs were confirmed by sequencing (Genewiz). cloned into RML6 vector and was expressed in CHO cells. Human ROMK truncation mutant (residues 35–367), mouse Constructs for Protein Purification. Mouse GIRK2 (mGIRK2) Kir2.1 truncation mutant (residues 41–368), and human Kir7.1 truncation mutant (residues 52–380) was cloned into RML1 vector truncation mutant (residues 13–360) were cloned into RML6 and was expressed in Pichia pastoris strain SMD1163 (Invitrogen). vector and were expressed in HEK293 cells. RML1 vector is modified from pPICZ (Invitrogen) by adding Mouse THIK1, mouse TRESK, rat TWIK2, mouse TALK1, a PreScission protease recognition sequence (LEVLFQ/GP) and mouse TASK3 genes were cloned into RML6 vector and were followed by an EGFP sequence and then a 1D4 tag sequence expressed in CHO cells. (TETSQVAPA) to the C terminus of cloned genes. Human Gβ1 and Gγ2 were individually cloned into pFastbac Protein Purification. vector (Invitrogen), and the Gβγ complex was expressed in High mGIRK2 purification. The cloned 1D4-tagged mGIRK2 construct was Five insect cells (Invitrogen) by virus coinfection. linearized with Pme1 and transformed into Pichia cells by elec- Mouse TRAAK (mTRAAK) truncation mutant (residues 1–275 troporation (Bio-Rad). Pichia cells were cultured in yeast extract with N81Q and N84Q glycosylation mutations) was fused to an peptone and dextrose (YPD) medium [1% yeast extract, 2% N-terminal 27 residue peptide from human TRAAK to increase (wt/vol) peptone, 2% (wt/vol) dextrose] in shakers (Infors HT; expression. Chimeric mouse TRAAK construct was cloned into Multitron) at 220 rpm and 30 °C. Transformants were selected by RML2 vector and was expressed in Pichia cells. RML2 vector is plating on standard YPD plate with 1 M sorbitol with 1 mg/mL modified from pPICZ by adding a PreScission protease recogni- zeocin (Invitrogen). Small-scale expression screens of individual tion sequence followed by an EGFP sequence and then a His10 clones were assessed by fluorescence-detection size-exclusion tag sequence to the C terminus of cloned genes. chromatography (40) (FSEC; Shimadzu CBM-20A). Before in- Human Slo1 (hSlo1) was cloned into RML11 vector and was duction, cells were cultured in a glycerol-based medium (1% expressed in Sf9 (Spodoptera frugiperda) cells (ATCC). RML11 NH4SO4, 0.34% yeast nitrogen base, 100 mM K-phoshate buffer, vector is modified from pFastBac vector by adding a PreScission pH 6.0, 1.6 μM biotin, 1% glycerol) at 220 rpm and 30 °C for 24 h. protease recognition sequence followed by an EGFP sequence Induction was initiated by switching to a methanol-based medium and then a 1D4 tag sequence to the C terminus of cloned genes. (1% NH4SO4, 0.34% yeast nitrogen base, 100 mM K-phosphate hERG truncation mutant with internal deletions of unstructured buffer, pH 6.0, 1.6 μM biotin, 0.5% methanol) at 27 °C and cytoplasmic loops (residues 141–380 and 871–1005) was cloned into continued for 24 h. The clone with the highest mGIRK2 expres- − RML13 BacMam vector and was expressed in HEK293S GnTI sion observed by FSEC was chosen for large-scale expression, cells (ATCC). RML13 vector is modified from pEG (39) (a generous performed in a fermenter (Infors HT Labfors). Overnight starter gift from Eric Gouaux, The Vollum Institute, Portland, OR), by cultures of cells grown in the glycerol-based medium with 1 mg/mL adding a PreScission protease recognition sequence followed by an zeocin were added to 3 L minimal media (containing 151.2 g EGFP sequence and then a 1D4 tag sequence to the C terminus of glycerol, 2.79 g CaSO4, 54.6 g K2SO4, 21.83 g MgSO4)toan ∼ cloned genes. OD600 1 and grown overnight at 29 °C with glycerol feeding and Three of the above constructs (for GIRK2, TRAAK, and pH maintained at 5.0 by addition of NH4OH. Cells were then hERG) represent channels that have been modified from their full- starved to deplete glycerol and temperature was reduced to 27 °C. length form to gain biochemical stability. Although it is possible Induction was initiated with slow addition of methanol and con- that disordered loops could form small molecule binding sites, the tinued for ∼24 h. Cells were pelleted, frozen in liquid nitrogen, + − function of GIRK2 (response to Na ,PIP2, and Gβγ,aswellasto and stored at 80 °C. toxin inhibitors) and TRAAK (mechanical sensitivity) are in- Frozen Pichia cells expressing mGIRK2 protein were dis- distinguishable from the full-length channels. For the case of the rupted by milling (Retsch; model MM301) for five cycles of hERG channel, the N-terminal truncation causes increased rates 3 min at 25 Hz. Cells were kept frozen between cycles by cooling of activation and deactivation. However, the correlation between in liquid nitrogen. All subsequent steps were performed at 4 °C. drug sensitivity between published electrophysiological assays us- Cell powder was added to lysis buffer [50 mM Hepes-KOH, ing the full-length channel and the LFA assay using the truncated pH 8.0, 150 mM KCl, 1 mM EDTA, 0.1 mg/mL DNase, a pro- channel lead us to believe that the truncated channel reports drug tease inhibitor mixture containing 1 μg/mL leupeptin, 1 μg/mL sensitivity with good fidelity. pepstatin, 1 μg/mL aprotinin, 100 μg/mL soy trypsin inhibitor, 1 mM benzamidine, 100 μg/mL 4-(2-aminoethyl) benzene- Constructs for Electrophysiology. mGIRK2 truncation mutant sulfonyl fluoride and 1 mM phenylmethysulfonyl fluoride] at a ratio (residues 1–414, for reference the full-length protein is 1–425) was of 1 g cell pellet/4 mL lysis buffer. After 1-h lysis with stirring, the cloned into RML6 vector and was expressed in HEK293 cells pH of the lysis buffer was readjusted to 8.0 using 1M KOH. Ex- (ATCC). RML6 vector is a mammalian cell expression vector traction was initiated by adding 60 mM n-Decyl-β-D-Maltopyrano- adding a thrombin protease recognition sequence (LVPRGS) side (DM, Anatrace) and continued for 1 h with stirring. Extracts followed by an EGFP sequence and then a His8 tag sequence to were collected by centrifugation at 35,000 × g (Beckman, Miami, the C terminus of cloned genes. Avanti J-26XP centrifuge, JA-17 rotor) for 30 min. Washed 1D4 Rat Kv1.2 was cloned into RML5 vector and was expressed in resin was added to the supernatant (1 mL resin/8 g cell) and binding CHO (ATCC) cells. RML5 vector is a mammalian cell expression continued for 1 h with rotation. After binding, resin was collected vector adding a His8 tag sequence followed by an EGFP sequence at 245 × g (Beckman, Miami, Avanti J-26XP centrifuge, JS-5.3

Su et al. www.pnas.org/cgi/content/short/1602815113 1of13 rotor) for 5 min. Resin was loaded onto a column and washed were extracted by gentle stirring for 3 h and collected by centri- with 20 column volumes (cv) of wash buffer (50 mM Hepes- fugation at 35,000 × g for 45 min. Washed TALON resin was KOH, pH 7.4, 150 mM KCl, 1 mM EDTA, 6 mM DM). PreScission added to the supernatant (1 mL resin/5 g cell pellet) and binding protease [∼1:20 (wt:wt)] was subsequently added to washed resin continued for 3 h with gentle stirring. Resin was loaded onto a and on-column cleavage continued for 1.5 h with gentle rotation. column and sequentially washed and eluted in wash buffer Cleaved protein was eluted in wash buffer, and 20 mM DTT was (50 mM Tris, pH 8.0, 150 mM KCl, 6 mM DDM) containing 10, added. Protein was concentrated [100-kDa molecular weight 30, and 300 mM imidazole. EDTA (1 mM final) and PreScission cutoff (MWCO), Amicon Ultra Centrifugal Filter] and applied protease [1:50 (wt:wt)] were added to the elution, and cleavage to a Superdex 200 column (GE Healthcare, 10/300 GL) equili- continued overnight with gentle rotation. Cleaved protein was brated in size exclusion chromatography (SEC) buffer (20 mM concentrated (50 kDa MWCO, Amicon Ultra Centrifugal Filter) Hepes-KOH, pH 7.4, 150 mM KCl, 1 mM EDTA, 20 mM DTT, and applied to a Superdex 200 column equilibrated in SEC buffer 4 mM DM). Peak fractions containing mGIRK2 protein were pooled (20 mM Tris, pH 8.0, 150 mM KCl, 1 mM EDTA, 1 mM DDM). andconcentrated(100kDaMWCO)to 1 mg/mL for reconstitution. Peak fractions containing mTRAAK protein were pooled and Human Gβγ purification. Individual baculoviruses expressing un- concentrated (50 kDa MWCO) to 1 mg/mL for reconstitution. tagged human Gβ1 and N-terminal His-tagged (followed by a hSLo1 purification. hSlo1 virus production followed the Bac-to-Bac PreScission Protease cleavable linker) human Gγ2 were pro- manufacturer’s instruction. Sf9 cells were cultured in Grace duced using the Bac-to-Bac system following manufacturer’s medium (Life Technologies) supplemented with 10% (vol/vol) instruction (Invitrogen). High Five cells were cultured in Express FBS, 1% pluronic F-68, and 1% penicillin/streptomycin in Five medium (Life Technologies) supplemented with 18 mM shakers (Infors HT, Multitron) at 120 rpm (Infors HT, Multi- L-glutamine and 1% penicillin/streptomycin in shakers (Infors tron) and 27 °C. Cells were infected with hSlo1 virus at a density of HT, Multitron) at 120 rpm and 27 °C. Cells were coinfected 2 × 106 cells/mL 60 h after infection, harvested by centrifugation, with Gβ1andGγ2 viruses (with a ratio Gβ1: Gγ2 = 2:1) at frozen with liquid nitrogen, and stored at −80 °C. a density of 2 × 106 cells/mL; 40 h after infection, cells were All steps were performed at 4 °C. Frozen Sf9 cells expressing harvested by centrifugation, frozen with liquid nitrogen, and hSlo1 protein were lysed and extracted in lysis buffer (50 mM stored at −80 °C. Hepes-KOH, pH 8.0, 150 mM KCl, 1 mM EDTA, a protease All steps were performed at 4 °C. Frozen High Five cells ex- inhibitor mixture the same as used in GIRK2 purification, 60 mM pressing Gβγ protein were lysed by sonication (Branson, Sonifier DM) for 2 h with stirring. Extracts were collected by centrifugation 450) for three cycles (maximum output, 15 s) on ice in lysis buffer at 257,000 × g for 40 min. Washed 1D4 resin was added to the (50 mM Hepes-NaOH, pH 8.0, 100 mM NaCl, 1 mM EDTA, supernatant (2 mL resin/1 L cell) and binding continued for 1 h 3 mM 2-mercaptoethanol, a protease inhibitor mixture containing with rotation. After binding, resin was collected at 245 × g for 1 μg/mL leupeptin, 1 μg/mL pepstatin, 1 μg/mL aprotinin, 5 min and washed briefly with wash buffer (50 mM Hepes-KOH, 100 μg/mL soy trypsin inhibitor, 1 mM benzamidine). Cells were pH 7.4, 150 mM KCl, 1 mM EDTA, 6 mM DM). Resin was loaded kept cold between cycles by cooling on ice. Cell lysates were to a column and further washed with20cvwashbuffer.PreScission centrifuged at 257,000 × g (Beckman, Miami, Optima XL-100K protease [∼1:30 (wt:wt)] was subsequently added to washed resin Ultracentrifuge, type 70 i rotor) for 40 min to collect membranes. and on-column cleavage continued for 1.5 h with gentle rotation. Membrane pellets were resuspended in resuspension buffer (50 mM Cleaved protein was eluted in wash buffer and 20 mM DTT was Hepes-NaOH, pH 8.0, 100 mM NaCl, 3 mM 2-mercaptoethanol, added. Protein was concentrated (100 kDa MWCO) and applied to a protease inhibitor mixture the same as in lysis buffer) and a Superose 6 column (GE Healthcare; 10/300 GL) equilibrated in homogenized in 40 mL Dounce Tissue Grinder (Wheaton). Ex- SEC buffer (20 mM Hepes-KOH, pH7.4, 150 mM KCl, 1 mM traction was initiated by adding 2% (wt/vol) Na-cholate (Sigma) EDTA, 20 mM DTT, 4 mM DM). Peak fractions containing and continued for 1 h with stirring. Extracts were collected by hSLO1 protein were pooled and concentrated (100 kDa MWCO) centrifugation at 35,000 × g for 30 min. Washed TALON resin to 1 mg/mL for reconstitution. (Clontech) was added to the supernatant (5 mL resin/1 L cell) and hERG purification. hERG virus production followed the BacMam − binding continued for 1 h with rotation. After binding, resin was virus protocol (39). HEK293S GnTI cells were cultured in collected at 245 × g for 5 min and washed briefly with wash buffer FreeStyle 293 Expression Medium (Life Technologies) supple- (50 mM Hepes-NaOH, pH 8.0, 100 mM NaCl, 3 mM 2-mercap- mented with 2% (vol/vol) FBS and 1% penicillin/streptomycin in toethanol, 1% Na-cholate). Resin was loaded to a column and incubators (Thermo; model 3950) equipped with tabletop orbital sequentially washed and eluted in wash buffer containing 0, 10, and shaker at 130 rpm (Thermo, MaxQ HP) and 37 °C with moisture 200 mM imidazole. PreScission protease [∼1:20 (wt:wt)] was sub- and 8% CO2. Cells were infected with hERG virus at a density of sequently added to eluted protein and cleavage continued for 1 h 3 × 106 cells/mL 24 h after infection, and 10 mM sodium butyrate with gentle rotation. Cleaved protein was concentrated (10 kDa (Sigma) was added to induce protein expression; 36 h after in- MWCO, Amicon Ultra Centrifugal Filter) and applied to a duction, cells were harvested by centrifugation, frozen with liquid Superdex 200 column equilibrated in SEC buffer (20 mM Hepes- nitrogen, and stored at −80 °C. − KOH, pH7.4, 150 mM KCl, 1 mM EDTA, 20 mM DTT, 4 mM All steps were performed at 4 °C. Frozen HEK293S GnTI cells DM). Peak fractions containing Gβγ complex were pooled and expressing hERG protein were lysed in hypo-osmotic lysis solution concentrated (100 kDa MWCO) to 30 mg/mL and frozen in (10 mM Hepes-NaOH, pH 7.4, 30 mM KCl, 5 mM DTT, 20 μg/mL liquid nitrogen at −80 °C for mGIRK2 reconstitution and LFA. DNase, a protease inhibitor mixture the same as used in GIRK2 mTRAAK purification. For mTRAAK purification, yeast cell trans- purification) by stirring to homogeny. Cell lysates were centrifuged formation, clone selection, and protein expression all followed the at 35,000 × g for 45 min to collect membranes. Membrane pellets protocol of mGIRK2 purification, except for that the methanol were resuspended in extraction buffer [20 mM Hepes-NaOH, induction time was 48 h in the fermenter culture. pH 7.4, 300 mM KCl, 20 μg/mL DNase, a protease inhibitor mixture Frozen Pichia cells expressing mTRAAK protein were dis- the same as used in GIRK2 purification, 1% DDM, 0.2% cholesteryl rupted by milling the same as mGIRK2 purification. All sub- hemisuccinate (CHS; Anatrace)], and extraction continued for 1 h. sequent steps were performed at 4 °C. Cell powder was added to Extracts were collected by centrifugation at 20,000 × g for 15 min. lysis buffer [50 mM Tris, pH 8.0, 150 mM KCl, 0.1 mg/mL DNase, Washed GFP-nanobody resin [CNBr-activated Sepharose 4B (GE a protease inhibitor mixture the same as used in mGRIK2 puri- Healthcare) conjugated with a GFP-binding nanobody (41, 42)] was fication, 60 mM n-dodecyl-β-D-maltopyranoside (DDM; Ana- added to the supernatant (5 mL resin/1 L cell), and binding con- trace)] at a ratio of 1 g cell pellet/4 mL lysis buffer. Membranes tinued for 1 h with rotation. After binding, resin was collected at

Su et al. www.pnas.org/cgi/content/short/1602815113 2of13 + 245 × g for 5 min and washed briefly with wash buffer [20 mM screening drug plate served multiple K channel assays, which Hepes-NaOH, pH 7.4, 300 mM KCl, 0.1% DDM, 0.02% CHS, reduces the cost of the screens. 0.1 mg/mL lipids [POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3- The primary screen. The primary screen was carried out with a phosphocholine (POPC): 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate FDSS6000 (Hamamatsu) plate reader with automatic pipetting in (POPA) = 5:5:1]. Resin was loaded onto a column and further 384-well format. Before screening, flat-bottomed 384-well plates washed with 20 cv wash buffer. PreScission protease [∼1:40 (wt:wt)] (Greiner Bio-One; cat. 781076) were filled with stock solutions of was added to washed resin and on-column cleavage continued for different components including drugs, ACMA, and CCCP. 1.5 h with gentle rotation. Cleaved protein was eluted in wash Vesicles were added to v-bottomed 384-well plates (Greiner Bio- buffer, concentrated (100 kDa MWCO), and applied to a Superose One; cat. 784201). Drug stock plates were filled with 2.5 μM 6 column equilibrated in SEC buffer [20 mM Hepes-NaOH, screening compounds in drug buffer (675 mM NaCl, 20 mM pH7.4, 300 mM KCl, 0.1% DDM, 0.02% CHS, 0.1 mg/mL lipids Hepes-NaOH, pH 7.4). Columns 23 and 24 were positive and (POPE:POPC:POPA = 5:5:1), 0.5 mM Tris(2-carboxyethyl)phos- negative control drugs, respectively. ACMA stock plates were μ phine (TCEP), 10 mM DTT]. Peak fractions containing hERG filled with 6.5 M ACMA in ACMA buffer (20 mM Hepes- μ protein were pooled for reconstitution. NaOH, pH 7.4). CCCP stock plates were filled with 16 M CCCP in a buffer (5 mM EDTA, 20 mM Hepes-NaOH, pH 7.4). Proteoliposome Reconstitution. Lipids in organic solvent were Vesicles containing different channel proteins were thawed in a handled with glass pipettes and vials; 10 mg/mL POPE and POPG 37 °C water bath for 30 min and briefly mixed to homogeny by [1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)] from gentle pipetting. Thawing at 37 °C is very critical in obtaining Avanti in chloroform were mixed [with a ratio of 3:1 (wt:wt)] and robust LFA signals. Homogeneous vesicles were left at room × temperature until immediately before the screening. Vesicles aliquoted in 4 mL per glass vial (Kimble Chase; 16 125 mm); 1% + were diluted into a buffer containing no K (150 mM NaCl, brain PIP2 lipid (Avanti) was added for the mGIRK2 inhibitor 20 mM Hepes-NaOH) before their addition to the stock plate to screen and the mGIRK2 PIP2-based activator screen. Lipid ali- + create a stronger K gradient across the vesicle membrane, en- quots were air-dried under argon, washed with pentane, and dried + hancing the flux signal. Vesicles were stable in high K solution again under argon in a laboratory chemical hood. Lipids were + for at least a day but gradually lost flux activity in low K high protected from light by wrapping with aluminum foil and protected + from oxidation by topping with argon in all subsequent steps. Na buffers, we therefore waited until right before the screen to dilute the vesicles. The vesicle dilutions used for screening are Lipids were further dried overnight in a vacuum chamber with – – loose caps on the vials. Each dried aliquot was rehydrated in 2 mL 1 40 for the mGIRK2 inhibitor screen, 1 30 for mGIRK2 PIP2- based activator screen, 1–60 for the mTRAAK inhibitor screen, reconstitution buffer (20 mM Hepes-KOH, pH7.4, 1 mM EDTA, – 150 mM KCl, except for hERG 300 mM KCl were used). Lipid and 1 30 for the hSlo1 activator screen. Vesicles in stock plates solutions were sonicated until translucent in a bath sonicator maintained their robust flux signals for at least 20 consecutive plates screened. Empty 384-well clear-bottom plates (Greiner (Laboratory Supplies; model G112SP1T) with multiple cycles of Bio-One; cat. 781096) were used as assay plates to mix flux 1 min on and 1 min off to prevent overheating. Sonicated lipid components and to read fluorescence. solutions were mixed with an equal volume of solubilization buffer Drug stock plates were loaded to the back port of FDSS6000, (reconstitution buffer with 20 mM DM (for mGIRK2, hERG) or β and empty assay plates were loaded to the front port. ACMA 160 mM n-octyl- -D-maltopyranoside [OM; Anatrace; for the other stock plates, vesicle stock plates, and CCCP stock plates were proteins) and 20 mM DTT (no DTT for mTRAAK)] and in- placed on the rotating stage automatically controlled by cubated for 30 min by rotating. Solubilized lipid-detergent solution FDSS6000. Pipettes loaded on the pipette header of FDSS6000 was mixed with purified protein at desired ratios and incubated for were automatically washed before and after each pipetting. All 1 h at room temperature by rotation. After incubation, protein- screens were conducted at room temperature except for the hSlo1 lipid-detergent solution was immediately transferred to 50 kDa activator screen, which was performed at 37 °C. MWCO dialysis bags (Spectrum Labs) in cold reconstitution buffer The FDSS6000 pipetted and mixed 12 μL drug solution, 6 μL with fresh 3 mM DTT (no DTT for mTRAAK) and dialyzed at ACMA solution, and 6 μL vesicle solution into an assay plate, 4°C(4Lbuffer/20mLprotein-lipid-detergent solution). Re- and the baseline fluorescence was recorded (using 380-nm ex- constitution buffer was changed every 12 h for six cycles. After citation and 510-nm emission). Then 6 μL CCCP was added and + visible vesicle formation, bags were massaged manually each day mixed to initiate the K flux, and the fluorescent signal was to break up large lipid aggregates. Prewetted Bio-Beads SM2 monitored every 5 s for 55 cycles. No valinomycin was used (Bio-Rad, first wetted in methanol, subsequently washed five during the primary screen because it adhered to the pipette tips times with ddH2O and one time with reconstitution buffer) and contaminated subsequent plates. The final concentrations were added to reconstitution buffer (5 mL/4 L buffer) for the for the components in the LFA reaction were 1 μM drug, 1.3 μM last three cycles to remove residual detergents; 50-μL aliquots ACMA, and 3.2 μM CCCP. of the proteoliposomes were flash-frozen in liquid nitrogen and For mGIRK2 inhibitor screen, the positive control was mGIRK2 − stored at 80 °C. vesicles reconstituted with PIP2 only, the negative control was mGIRK2 reconstituted with both Gβγ and PIP2, and the Z-factor High-Throughput LFA Screens. was 0.84. For mGIRK2 activator screen, the positive control was Compound library collections. Three hundred thousand compounds DM-solubilized Gβγ, the negative control was DM only, and the were collected for screening, of which 100,000 were from En- Z-factor was 0.81. For mTRAAK inhibitor screen, the positive amine (a preplated diverse subset of their library), 100,000 were control was a nonspecific pore blocker in the beginning and RU- from ChemBridge (DIVERSet-EXP and DIVERSet-CL), and TRAAK-1 for the rest of the screen, the negative control was 100,000 were from the library available at The Rockefeller DMSO only, and the Z-factor was 0.91. For hSlo1 activator + University High-Throughput and Spectroscopy Resource Center screen, the positive control was Ca2 in the beginning and RU- (RU-HTSRC). The compounds were selected to maximize di- Slo1-2 for the rest of the screen, the negative control was DMSO versity. These compounds were stored frozen in DMSO at −20 °C only, and the Z-factor was 0.82. in 384-well stock plates at the concentration of 5–10 mM in al- Hit confirmation using LFA. Once the primary screens were finished iquots. On the day of screening, drug DMSO plates were thawed and primary hits were selected using offline data analysis, the hits and small volume of drugs were pipetted out and diluted into were confirmed using a fluorescent plate reader (Tecan, Infinite screening drug plates filled with drug buffer by robot. The same M1000 with excitation wavelength 410 nm and emission wavelength

Su et al. www.pnas.org/cgi/content/short/1602815113 3of13 490 nm) with manual pipetting following the same protocol as the and S2, respectively, the offset does not render the assay inac- primary screen in 384-well plates. After monitoring the flux signal curate in its ability to identify channel blocking agents. for 55 cycles, 1 μL8μM valinomycin (in DMSO) was added to allow + K ions to pass through the membrane and reach equilibrium. The Animals. All animal protocols were approved by IACUC of The final fluorescence value was then recorded. Valinomycin addi- Rockefeller University. Newborn C57BL/6J mice (The Jackson tion causes little change in ACMA fluorescent signal under Laboratory) were killed for the dissection of hippocampal neurons. conditions where the channels are active, but it causes a sharp Hippocampal Neuron Cultures. Twelve-millimeter round poly-D-lysine– drop in signal under conditions where the channels are blocked coated glass coverslips (neuVitro) were further coated with 10 μg/mL or inactive. The use of valinomycin is an important control be- laminin (Invitrogen) in Dulbecco’s PBS buffer (DPBS; Life cause fluorescent compounds may be identified as inhibitors in Technologies) at 37 °C for 3 h in 24-well plates (Falcon). Before the primary screen, but these are likely false positives if the dissection, coated coverslips were washed four times with DPBS fluorescent signal does not decrease after valinomycin addition. and incubated with plating medium (Neurobasal-A medium; Life Similarly, compounds that lyse the vesicles may show up as false Technologies) supplemented with 1× B27 supplement, 2% positives in the primary screen, and can be discarded after an (vol/vol) horse serum, 2 mM GlutaMAX supplement, and 25 μM anomalous postvalinomycin result. glutamate at 37 °C with moisture and 5% CO2. Hippocampus Once the hits were confirmed, fresh compounds were pur- tissues from P0 mice were quickly dissected out in cold Hank’s + chased from the same vendors that had supplied the primary balanced salt solution (HBSS; Life Technologies) without Ca2 + screening compounds and drug titrations were conducted. Each and Mg2 . Hippocampus tissues were digested with 0.25% compound titration was performed in a single column of a 384- trypsin (Sigma; 1:250 tryptic activity) at 37 °C for 15 min. Di- well plate to ensure the simultaneous addition of CCCP using a gested tissues were washed four times with warm HBSS without 2+ 2+ multichannel pipette in a Tecan Infinite M1000 plate reader. The Ca and Mg and gently triturated by a P200 pipette. Viable flux data were normalized, plotted, and fitted as detailed later. cells were counted with Trypan Blue (Lonza) and plated on coated coverslips in plating medium at a density of 0.5–1 × 105 Explanation of the IC50 Offset in the hERG Assay. In the LFA, efflux cells per well. Four days after plating, plating medium was + + of K is coupled to ionophore-mediated influx of H . Through gradually changed to culturing medium (plating medium without analysis of efflux rates as a function of protein-to-lipid ratio with glutamate). Culturing medium was renewed every 3 d. Neurons + various K channels, we know that under many circumstances matured after 1 wk and were patched between 1 and 2 wk after (specific channels such as hERG and at high protein-to-lipid plating. + + ratios), when K channels are not blocked, the influx of H limits + + the efflux of K . Consequently, reduced K efflux is observed Cell Line Cultures. HEK293 cells were cultured in DMEM (Life + + Technologies) supplemented with 10% (vol/vol) FBS, 2 mM only when K channels are sufficiently blocked to allow K efflux L-glutamine, and 1% penicillin/streptomycin in an incubator to become rate limiting. This effect causes an offset in the esti- + (Thermo Forma; Series II 3110) at 37 °C with moisture and mate of the IC50 of a K . To demonstrate with a + 5% CO . CHO-K1 cells were cultured in DMEM: Nutrient simple model how this occurs, we model the processes of K 2 + Mixture F-12 (DMEM/F12; Life Technologies) supplemented efflux and H influx as conductors in series: GK and GH. with 10% (vol/vol) FBS, 2 mM L-glutamine, and 1% penicillin/ Therefore, recorded conductance is streptomycin in an incubator at 37 °C with moisture and 5% − CO2. Cells were cultured to 80% confluency and transfected = −1 + −1 1 [S1] Grec GK GH . with Lipofectamine 2000 (Invitrogen) following the manufac- turer’s protocol and passaged 12–24 h after transfection onto + If a blocker at concentration x inhibits K conductance according to 12-mm poly-D-lysine– and laminin-coated glass coverslips (BioCoat) at low density. Patch-clamp recordings were performed 6–48 h x after plating. G = G × 1 − , [S2] K K0 K + x d Electrophysiology. All recordings were performed at room tem- + perature. Pipettes of borosilicate glass (Sutter Instruments; BF150- where G is the K conductance in the absence of blocker, then K0 86-10)werepulledto2-to4-MΩ resistance (except the pipettes – Ω −1 −1 used to patch BK were 1 2M ) with a micropipette puller (Sutter − = −1 + −1 1 = × − x + −1 Instruments; P-97) and polished with a microforge (Narishige; Grec GK GH GK0 1 + GH , Kd x MF-83). Recordings were made with an Axopatch 200B amplifier [S3] (Molecular Devices) using standard whole-cell or excised patch- clamp techniques. Current-clamp experiments were performed on where Kd is the equilibrium dissociation constant for the blocker the I-fast mode. Recordings were acquired at 10 kHz and filtered binding to its inhibitory site on the channel. Fig. 5G shows that a at 1 kHz (Molecular Devices; 1440A). Pressure stimulation in titration curve is shifted ∼10-fold when GK0 = 10 GH. By setting inside-out patches for TRAAK was performed with a high-speed . pressure clamp (ALA Scientific; HSPC) controlled through the −1 G = G−1 + G−1 2, [S4] Clampex 10 software (Molecular Devices). Pressure application rec K0 H velocity was set to the maximum rate of 8.3 mmHg/ms. For whole-cell recordings of the mGIRK2 channel for inhibitor we find that the IC50 for the LFA curve is characterizations, the pipette solution was 10 mM Hepes-KOH, 20 mM NaCl, 130 mM KCl, 5 mM EGTA, and 2 mM MgCl2, GK0 pH 7.4 (adjusted with KOH), and the bath solution was 10 mM IC50 = + 1 × Kd. [S5] GH Hepes-KOH, 150 mM KCl, 5 mM EGTA, and 2 mM MgCl2, pH 7.4 (adjusted with KOH). For whole-cell recordings of the This model explains in simple terms why LFA can contain an off- mGIRK2 channel with ivermectin, symmetrical pipette and bath set in the estimated affinity of a blocker. As shown by the absence solutions were 10 mM Hepes-KOH, 150 mM KCl, 5 mM EGTA, of false-negative and false-positive determinations in Tables S1 and 2 mM MgCl2, pH 7.4 (adjusted with KOH). For whole-cell

Su et al. www.pnas.org/cgi/content/short/1602815113 4of13 recordings of the Kv1.2, hKir1.1, mKir2.1, hKir7.1, or K2P chan- and Fend is the measured end point fluorescence after addition of nels, the pipette solution was 10 mM Hepes-KOH, 150 mM KCl, valinomycin. Normalizations were performed with Excel (Micro- 5 mM EGTA, and 2 mM MgCl2, pH 7.4 (adjusted with KOH), soft). Plots were made using Prism software (GraphPad). and the bath solution was 10mM Hepe-NaOH, 15 mM KCl, The initial slopes of flux after CCCP addition (the average 140 mM NaCl, 2 mM MgCl2,and1mMCaCl2, pH 7.4 (adjusted slopes of first 10 s after CCCP addition) were extracted from with NaOH). For inside-out recordings of the mTRAAK channel, normalized flux titration data. The slope values were normalized to the pipette solution was 10 mM Hepe-NaOH, 15 mM KCl, the maximum slope to obtain normalized flux activity plotted in the 140 mM NaCl, 2 mM MgCl2,and1mMCaCl2, pH 7.4 (adjusted dose–response curves of flux titrations. Of note, in an inhibitor with NaOH), and the bath solution was 10 mM Hepes-KOH, titration, the maximum slope is the slope of DMSO control 150 mM KCl, 5 mM EGTA, and 2 mM MgCl2, pH 7.4 (adjusted whereas in an activator titration the maximum slope is the slope at with KOH). For whole-cell and inside-out recordings of the hSlo1 highest activator concentration tested. The dose–response curves channel, symmetrical pipette and bath solutions were 10 mM of flux titrations were fitted with the Hill equation with OriginPro Hepes-KOH, 150 mM KCl, 5 mM EGTA, and 2 mM MgCl2, software (OriginLab) pH 7.4 (adjusted with KOH). For whole-cell hippocampal neuron recordings, the pipette solution was 10 mM Hepes-KOH, 150 mM xn y = START + ðEND ‒ STARTÞ [S7] K-gluconate,5mMNaCl,5mMEGTA,2mMMgCl2,4mM kn + xn Mg-ATP, 0.4 mM Na-GTP, and 10 mM phosphocreatine (Tris salt), pH 7.4 (adjusted with KOH), and the bath solution was 10 mM where y is the normalized flux activity (the normalized initial Hepes-NaOH,140mMNaCl,5mMKCl,2mMMgCl2,2mM slope), START is the normalized flux activity of DMSO control, CaCl2, and 10 mM glucose, pH 7.4 (adjusted with NaOH). END is the normalized flux activity at the highest drug concen- All drug perfusions were performed using home-made bath tration tested, x is drug concentration, and n is the Hill coeffi- perfusion chambers except for the hippocampal neuron record- cient. The Hill coefficient ranged between 0.8 and 3.2. ings, which used a fast pressurized microperfusion system (ALA Current-voltage curves of Slo1 recordings (Fig. 4D and Fig. S5) Scientific; ALAVC3X8PP). were fitted with the Boltzmann equation in Prism

Data Analysis. All data are presented as mean ± SEM. Itop − Ibottom I = I + − , [S8] bottom ZF ðV−V Þ Flux titration data were first normalized to eliminate baseline 1 + e RT mid fluorescence fluctuations (due to ACMA pipetting variance and intrinsic fluorescence of testing compounds) using the following where I is the measured current, Ibottom is the measure current at equation: the lowest voltage, Itop is the measure current at the highest voltage, V is the command depolarization voltage to open the − F Fend channels, Vmid is the command voltage at which the channels Fnormalized = , [S6] Fstart − Fend have reached halfway between Ibottom and Itop, F is the Faraday constant, R is the gas constant, T is the absolute temperature, where Fnormalized is the normalized fluorescence plotted in the flux and Z is the apparent valence of the voltage dependence. titration figures, F is measured fluorescence in arbitrary units, Fstart The scatter plot of hERG positive control drugs (Fig. 5F) was is the average of measured fluorescence before addition of CCCP, fitted with linear regression in OriginPro (OriginLab).

A B C mGIRK2 protein: lipid titration C8-PIP titration DM-solubilized Gβγ titration 1.0 1.0 2 1.0

0.5 0.5 (μM) 0.5 mg/ml empty 0 0 1 1: 106 0.1 5 2 0.2 1: 10 5 1 1: 104 10 2 3 Normalized fluorescence 0 Normalized fluorescence Normalized fluorescence 0 1: 10 20 0 5 0 100 200 300 0 100 200 300 0 100 200 300 Time (s) Time (s) Time (s)

Fig. S1. Optimization of mouse GIRK2 screens. (A) Protein-lipid ratio titrations of GIRK2 reconstituted with both brain PIP2 and lipidated Gβγ. Low protein 4 5 lipid ratio ∼1: 10 -1:10 produced robust flux signals sensitive to blockage and ideal for inhibitor screening. (B) C8-PIP2 titration of GIRK2 reconstituted with lipidated Gβγ only. In the absence of brain PIP2, GIRK2, and Gβγ containing vesicles showed little flux. Additions of soluble short-chain C8-PIP2 induced robust flux by activating the channels whose cytoplasmic domain face the outside of the vesicles. (C) DM-solubilized Gβγ titration of GIRK2 reconstituted with brain-

PIP2 only. Without lipidated Gβγ, GIRK2 and brain-PIP2 containing vesicles showed weak flux. Addition of DM-solubilized Gβγ evoked robust flux by activating inside-out facing channels.

Su et al. www.pnas.org/cgi/content/short/1602815113 5of13 A B RU-GIRK-2 RU-GIRK-2 titration I (pA) 1.0 1.0 N -100 -50 N 50 N (μM) V (mV) O DMSO 0.01 0.5 0.5 RU-GIRK-2 0.03 -500 0.1 (μM) 0.3 0 1 10 3 Normalized flux activity

Normalized fluorescence 0 O 0 10 IC50 = 0.534 + 0.124 μM -1000 N 0 100 200 300 0.001 0.01 0.1 1 10 N Time (s) [RU-GIRK-2] (μM) N C D RU-GIRK-3 RU-GIRK-3 titration I (pA) 1.0 1.0 -100 -50 N N 50 (μM) V (mV) N DMSO 0.03 -1000 RU-GIRK-3 0.5 0.1 0.5 O O O (μM) 0.3 0 1 10 O 3 -2000 10 Normalized flux activity Normalized fluorescence 0 30 0 IC50 = 0.916 + 0.193 μM 0 100 200 300 0.001 0.01 0.1 1 10 Time (s) [RU-GIRK-3] (μM) F E RU-GIRK-4 RU-GIRK-4 titration I (pA) 1.0 1.0 -100 -50 50 (μM) V (mV) DMSO -500 0.5 0.01 0.5 RU-GIRK-4 0.03 (μM) 0.1 0.3 -1000 0 1 10 NH2

3 Normalized flux activity 0 Normalized fluorescence 0 IC50 = 0.475 + 0.075 μM 10 -1500 0 100 200 300 0.001 0.01 0.1 1 10 Time (s) [RU-GIRK-4] (μM) G H 100 100 100 100

80 80 80 80

60 60 60 60 40 40 40 40 20 μM inhibitor in patch 20 20 20 μM RU-GIRK-3 in flux μM RU-GIRK-4 in flux 0 Percentage inhibition (%) at 10 Percentage inhibition (%) Percentage inhibition (%) 0 Percentage inhibition (%) 0 0 at 10 at 10 μM RU-GIRK-2 in flux at 10

Slo1 Slo1 Slo1 GIRK2 GIRK2 GIRK2 TRAAK TRAAK TRAAK RU-GIRK-2RU-GIRK-3RU-GIRK-4

Fig. S2. Characterization of other GIRK2 inhibitors. (A, C, and E) Chemical structures, normalized flux titrations, and curve fitting (Hill function) of dose– response curves of other GIRK2 inhibitors (n = 9 for RU-GIRK-2, n = 5 for RU-GIRK-3, and n = 9 for RU-GIRK-4). (B, D, and F) Representative whole-cell recordings of GIRK2 expressed in HEK293 cells in response to other GIRK2 inhibitors (n = 3 each). Pipette solution contained 20 mM Na+ to potentiate basal current and 10 μM drugs were applied. (G) Quantifications of recordings in B, D, and F (n = 3). (H) Selectivity test of GIRK2 inhibitors on different K+ channels using LFA (n = 3 each). All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 6of13 A Ivermectin titration EC50 = 1.6 + 0.2 μM 1.0 1.0

(μM) DMSO 0.5 0.1 0.5 0.3 1 2 5 Normalized flux activity Normalized fluorescence 0 10 0 0 100 200 300 0.01 0.1 1 10 Time (s) [Ivermectin] (μM) B Ivermectin (μM) 0 1 2 5 10 10 + TPNQ

500 pA

100 ms

C D I (pA) -100 -50 1.0 50 V (mV)

-500 Ivermectin 0.5 (μM) 0 -1000 1 2 Normalized current 5 0 10 M M -1500 10 + TPNQ 0 μ 10 μ TPNQ

M +

10 μ

Fig. S3. Characterization of a mouse GIRK2 activator, ivermectin (IVM). (A)(Left) GIRK2 was reconstituted with brain PIP2 only and addition of IVM induced robust flux, short-circuiting the need for Gβγ.(Right) Dose–response curve of GIRK2 flux with IVM (n = 3). (B) A representative whole-cell recording of GIRK2 + expressed in HEK293 cells in response to IVM (n = 3). Pipette solution contained no Na and the basal activities of GIRK2 was low. Addition of IVM to the bath activated large inward currents which were sensitive to TPNQ, indicating the currents being GIRK2 in origin. Membrane voltage was held at 0 mV, stepped from −120 to +40 mV in 20-mV increments, and returned to 0 mV. (C) Current-voltage plot from recordings in B.(D) Summary of IVM activation; 10 μM IVM- activated current was normalized to 1. All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 7of13 ihwoecl ac lm ( clamp patch whole-cell with a eesr ooti outfu inlta srsosv oihbtr.( inhibitors. to responsive is that signal flux robust a obtain to necessary was i.S4. Fig. ue al. et Su el nteasneadpeec f10 of presence and absence the in cells ( TRAAK-2. www.pnas.org/cgi/content/short/1602815113 os RA lxotmzto n hrceiaino h eodTAKihbtr ( inhibitor. TRAAK second the of characterization and optimization flux TRAAK Mouse Right Dose ) – epnecreo RA lxi h rsneo UTAK2( RU-TRAAK-2 of presence the in flux TRAAK of curve response B A C n -100 RU-TRAAK-2

= Normalized fluorescence 0.5 1.0 mTRAAK protein:lipidtitration ah.Aldt r mean are data All each). 3 0 (μM) 0 0 300 200 100 0 S 10 0 -50 N empty 1: 10 1: 10 1: 10 1: 10 I (pA) μ 300 RU-TRAAK-2 UTAK2( RU-TRAAK-2 M 4 5 6 7 N O H N 50 V (mV) 100 Time (s) n 400 = ± ) ( 3). D SEM.

D Percentage inhibition (%) UTAK2ihbto fdfeetK different of inhibition RU-TRAAK-2 ) at 10 μM RU-TRAAK-2 in patch 100 Normalized fluorescence 0.5 1.0 50 0 0

K 300 200 100 0 v 1.2 10 5 2 1 0.5 0.2 0.1 DMSO (μM)

B Slo1 RU-TRAAK-2 titration

)( GIK2 Left

UTAK2ceia tutr.( structure. chemical RU-TRAAK-2 ) TRAAK n

= THIK1

) ( 6). TRESK Time (s) TWIK2 C hl-elcretvlaecre rmTAKepesn CHO expressing TRAAK from curves current-voltage Whole-cell ) A TALK1 rti-ii-ai irto eeldta o ai ( ratio low a that revealed titration Protein-lipid-ratio ) TASK3

Normalized flux activity 0.5 1.0 0.01 0 + IC50 =0.719+0.079 hnesepesdi utrdclsadrecorded and cells cultured in expressed channels [RU-TRAAK-2] (μM) 0.1 Center 1 μ omlzdfu irto fRU- of titration flux Normalized ) M 10 ∼ 8of13 1:10 6 ) Fig. S5. Characterization of other human BK activators. (A, C, E, G, I, and K) Chemical structures and normalized efflux curves in the presence of Slo1 activators (n = 9 for RU-Slo1-2, n = 6 for RU-Slo1-3, n = 6 for RU-Slo1-4, n = 3 for RU-Slo1-5, n = 6 for RU-Slo1-6, n = 5 for RU-Slo1-7). (B, D, F, H, J, and L) Current-voltage plots from whole-cell patch recordings of Slo1 activators (n = 3 each). All activators induced leftward shifts to different degrees, indicating activation of Slo1 at less depolarizing voltages. All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 9of13 Fig. S6. Characterization of hERG positive control drugs. (A–D) Efflux curves and dose–response curves of well-characterized hERG blockers, E-4031, , , and (n = 3 each). (E–G) Efflux curves and dose–response curves using drugs that were withdrawn from the market because of risk of serious cardiac arrhythmias and increased risk of sudden death. These drugs have been shown to inhibit hERG activities from electrophysiological recordings. The LFA IC50 values correlate well to electrophysiological recordings with an approximate 10-fold IC50 offset. All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 10 of 13 lsammrn eengtv ntehR flxasya 10 at assay efflux hERG the in negative were membrane plasma elpaemitiigalcmoetrto.Tekntc n niioyac inhibitory and kinetics The ratios. component all maintaining plate well S8. Fig. 100 of concentration a at LFA in efflux hERG-mediated inhibit not did hERG, S7. Fig. ue al. et Su www.pnas.org/cgi/content/short/1602815113 EGmdae flxi ,3-elpae.Dftld a sda etp test a as used was plates. 1,536-well in efflux hERG-mediated aiaino EGngtv oto rg.( drugs. control negative hERG of Validation HI GH F DE C AB 1.0 0.5 0.5 1.0 Normalized fluorescence Normalized 0.5 fluorescence1.0 Normalized fluorescence 0 0 0 0 0 300 200 100 0 0 0 300 200 100 0 0 0 300 200 100 0 100 μM 100 μM 100 μM DMSO DMSO DMSO Salbutamol Ivermectin Time (s) Time (s) Time (s) A – D Dofetilide titrationin1536-wellplates

Normalized fluorescence inhibit to not known are that salbutamol, and ampicillin, HMR1556, aspirin, drugs, used widely Four ) 0.5 1.0 0 0 200 100 0 0.5 1.0 0.5 1.0 1.0 Normalized fluorescence Normalized fluorescence Normalized 0.5 fluorescence μ 0 0 0 1 0.3 0.1 0.03 0.01 0.003 0.001 DMSO Glaayi)ad100 and (Geldanamycin) M (μM) 0 0 300 200 100 0 0 0 300 200 100 0 0100200300 100 μM 100 μM DMSO DMSO DMSO 10 μM iiyo oeiiei h 56wl lt a iia ovle sn th using values to similar was plate 1536-well the in dofetilide of tivity Geldanamycin Bufexamac HMR1556 μ stv rg h lxrato ouewsrdcdt ure fta sdi used that of quarter a to reduced was volume reaction flux The drug. ositive M( n = Time (s) Time (s) Time (s) ah.( each). 3 Time (s) μ E ohr)( (others) M – 300 I iedusta r eotdt nii EGtafcigto trafficking hERG inhibit to reported are that drugs Five ) 0.5 1.0 0.5 1.0 Normalized fluorescence Normalized 0.5 fluorescence1.0 Normalized fluorescence 0 0 0 0 0 300 200 100 0 0100200300 0100200300 100 μM 100 μM 100 μM DMSO DMSO DMSO n = Pentamidine Ethacrynic acid each). 3 Ampicillin Time (s) Time (s) Time (s) 8 wells. 384 e 1o 13 of 11 384- a n Table S1. IC50 values of 50 hERG positive control drugs determined using LFA and compared with electrophysiology-determined values reported in the literature (n = 3 each) hERG positive controls Target IC50 patch (μM) IC50 flux (μM)

Amiodarone Class III antiarrhythmic 0.015–1.071 0.447 ± 0.029 Tricyclic 1.7–3.0 12.531 ± 1.320 Amsacrine Antineoplastic 0.209–0.230 1.208 ± 0.036 Class 1b antiarrhythmic 0.23 1.881 ± 0.221 0.001–0.018 0.074 ± 0.005 Class ΙΙΙ antiarrhythmic 0.891 4.275 ± 1.027 blocker 0.023–0.099 0.595 ± 0.066 Dopamine antagonist 0.37 3.179 ± 0.297 Gastroprokinetic agent 0.007–0.091 0.237 ± 0.014 Antihistamine 0.012 0.323 ± 0.018 Clomiphene Estrogen 0.18 4.582 ± 0.598 Cloperastine Cough suppressant 0.027 0.796 ± 0.119 Clotrimazole Antifungal 1.13 12.774 ± 0.304 Antipsychotic 0.468 7.451 ± 0.308 Dofetilide Class III antiarrhythmic 0.003–0.015 0.038 ± 0.002 Anti-dopamine 0.162 0.635 ± 0.069 Doxazosin Alpha blocker 0.6 5.128 ± 0.797 Antidopaminergic 0.100–0.307 0.806 ± 0.064 E-4031 Class III antiarrhythmic 0.008–0.134 0.047 ± 0.003 NMDA antagonist 0.02 0.110 ± 0.009 Serotonin reuptake inhibitor 2.6 11.005 ± 0.959 Antidepressant 0.500–0.720 4.179 ± 0.237 Antipsychotic 0.003 0.164 ± 0.010 GBR-12909 Dopamine reuptake 0.007 0.233 ± 0.022 Halofantrine Antimalaria 0.022–0.197 0.376 ± 0.018 Antipsychotic 0.015–0.063 0.178 ± 0.014 Ibutilide Class III antiarrhythmic 0.02 0.035 ± 0.002 NMDA antagonist 0.41 1.589 ± 0.071 1.9 14.934 ± 1.618 KB-R7943 Na/Ca exchanger inhibitor 0.089 1.775 ± 0.140 Antihypertensive 0.121–0.128 1.590 ± 0.247 Antifungal 1.9 19.653 ± 2.394 0.017–0.037 0.225 ± 0.021 Tetracyclic 3.1–5.2 4.882 ± 0.314 Antimalaria 2.6–5.6 2.969 ± 0.421 Piperidine neuroleptic 0.426 6.647 ± 1.393 Ca channel blocker 1.4 2.650 ± 0.141 Pimozide Antipsychotic 0.001–0.018 0.081 ± 0.014 Class I antiarrhythmic 0.320–1.5 5.520 ± 1.254 Antipsychotic 0.282 4.317 ± 0.447 Antipsychotic 0.003–0.210 0.387 ± 0.015 Tamoxifen Estrogen receptor 0.777 11.874 ± 0.678 Antihistamine 0.007–0.204 0.927 ± 0.036 Antispasmodic 0.375–0.700 9.640 ± 1.592 Antipsychotic 0.116–1.3 2.311 ± 0.160 Antimuscarinic 0.017 0.166 ± 0.008 Trazadone Antidepressant 0.690–2.9 9.248 ± 0.529 Antipsychotic 0.234 0.734 ± 0.078 Verapamil Calcium channel blocker 0.143–2.6 4.250 ± 0.753 Antipsychotic 0.120–0.240 4.061 ± 0.475

Measured IC50 values were plotted against the upper limit of IC50 values in electrophysiology in Fig. 5F.No false-negative drugs were found. All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 12 of 13 Table S2. Percent blockage of 50 hERG negative drugs determined by LFA Percentage inhibition (%) hERG negative controls Target at 100 μM

Acetaminophen Pain 11.6 ± 7.5 Acetazolamide Carbonic anhydrase inhibitor 4.4 ± 1.8 Antihistamine 16.2 ± 5.7 Enac channel blocker 15.4 ± 1.0 Amoxillin Antibiotic −2.6 ± 9.1 Ampicillin Antibiotic 5.6 ± 4.7 Arterenol Hormonre and neurotransmitter 3.7 ± 4.3 Aspirin Pain −1.6 ± 2.4 Bufexamac Anti-inflammatory 1.7 ± 6.8 Captopril ACE inhibitor 1.8 ± 2.9 receptor 2.2 ± 4.2 Antihistamine 11.8 ± 4.3 Antihistamine 19.9 ± 3.7 Clindamycin Antibiotic 9.2 ± 4.3 Clonidine Alpha2 agonist 15.2 ± 5.8 N-oxide DREADD agonist 2.1 ± 3.0 Doxycycline Antibiotic 1.6 ± 8.7 Enalapril ACE inhibitor −0.3 ± 6.8 Ethacrynic acid Loop diuretics 8.1 ± 4.4 Antihistamine −11.1 ± 15.8 Hypertension and edema −3.7 ± 16.1 Geldanamycin Antitumor antibiotic −1.5 ± 2.3** (10 μM) Glyburide Antidiabetic 4.0 ± 12.0 Expectorant −3.5 ± 7.4 HMR-1556 KCNQ1 blocker 6.5 ± 6.9 Anti-inflammatory 13.1 ± 2.1 Indapamide Diuretic 4.8 ± 6.7 Ivermectin Antiparasitic −21 ± 2.9 Kynurenic acid Antiexcitotoxic −8.3 ± 4.8 Nav blocker 13.0 ± 2.8 Midodrine Vasopressor 8.2 ± 4.8 Minocycline Antibiotic −2.5 ± 11.9 N-acetylprocainamide Class III antiarrhythmic 14.6 ± 4.2 Naproxen Cyclooxygenase inhibitor 3.8 ± 9.9 Oxypeucedanin Anti-tumor 4.5 ± 19.3 Penicillin Antibiotic −0.4 ± 9.5 Pentamidine Antimicrobial 22.8 ± 1.4 Phenylephrine α1-adrenergic receptor agonist 0.7 ± 5.9 Picrotoxin GABAA channel blocker 3.3 ± 4.0 Pyridoxine Vitamin B6 −5.0 ± 3.7 Antihistamine 14.9 ± 2.9 Resveratrol Natural phenol 21.1 ± 8.6 Salbutamol Beta2 agonist −6.6 ± 3.3 Spiramycin Antibiotic −5.8 ± 10.7 Sulfamethoxazole Antibiotic 11.5 ± 4.3 Sulindac Anti-inflammatory 1.1 ± 2.8 Thalidomide Immunomodulatory 9.5 ± 8.1 Trimethoprim Antibiotic 20.6 ± 1.9 Warfarin Anticoagulant 9.1 ± 5.5 Wortmannin PI3K inhibitor 1.7 ± 5.8

Drugs were tested at a concentration of 100 μM except for geldanamycin (**), which was tested at 10 μMdueto its auto fluorescence (n = 3 each). No drugs induced more than 25% difference compared with DMSO controls at this high concentration and were all considered negative. No false-positives were found. All data are mean ± SEM.

Su et al. www.pnas.org/cgi/content/short/1602815113 13 of 13