Supporting Information

Supporting Information 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.

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