Supporting Information

Tilley et al. 10.1073/pnas.1406876111 SI Materials and Methods CGSGKPACCP KYVCSPKWGL CNFPAPDLGT DDDDK, Peptide Synthesis and Folding. All mutants were made in a back- m/z = 6098.8). ground where the methionine at position 35 of guangxitoxin-1E + was replaced by its isostere norleucine to avoid any complications Toxin Conjugation. Acm deprotection was achieved with TFA of methionine oxidation. Norleucine35 variants are used in all 1% anisole for a final concentration of 10 mg/mL (2.5 mM) experiments, and referred to as GxTX. Linear peptides were GxTX. Silver acetate was added to 30 mg/mL (0.18 M), pro- synthesized on an AAPTEC Apex 396 peptide synthesizer using tected from light, mixed via slow rotation for 2 h, and monitored an Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology. Pep- by MALDI-TOF. Peptides were pelleted with diethyl ether as tides were assembled stepwise on 0.1–0.3 mmol resin (Fmoc-Pro- above. The remaining silver was removed by dissolving the pellet NovaSyn TGT, Novabiochem) in N-methyl-2-pyrrolidone, 0.4 M in 50% (vol/vol) AcOH, adding 1 volume of 2 M guanidinium Fmoc amino acids, 0.44 M N-hydroxybenzotriazole, and 10% HCl, incubating for 10 min in room temperature, and pelleted by (vol/vol) N,N′-diisopropylcarbodiimide. The side chain protect- centrifugation. The resultant GxTX Cys13 peptide was purified by HPLC as above. GxTX Cys13 was labeled with a maleimido– ing groups for amino acids were triphenylmethyl or acetamido- fluorophore, either tetramethylrhodamine maleimide (Life Tech- methyl (Acm) for cysteine and asparagine, tert-butyloxycarbonyl nologies T6027) or DyLight 550 Maleimide (Thermo 62290), to for tryptophan and lysine, and tert-butyl for serine. Removal yield GxTX-TMR or GxTX-dy550, respectively. GxTX Cys13 of Fmoc groups with 20% 4-methylpiperidine in dimethylform- lyophilisate was brought to 300 μM in 50% (vol/vol) ACN + 1 amide (DMF) preceded 2-h coupling steps. Resin was washed mM Na EDTA. One part 200 mM Tris, 20 mM Na EDTA (pH five times with DMF after coupling and Fmoc removal. Linear 2 2 6.8 with HCl) was mixed with this solution. A 2.5-mM solution of peptides were cleaved and deprotected with trifluoracetic the maleimido–fluorophore in DMSO was added to 20% (vol/vol) acid (TFA): triisopropylsilane:1,2-ethanedithiol:thioanisole:H2O – final DMSO concentration. The reaction was agitated in a poly- (85:2.5:2.5:5:5 by volume) for 2 4 h at room temperature, with propylene tube for 2 h at 20 °C. The conjugate was purified by removal of deprotecting groups monitored by MALDI-TOF HPLC, as above with a C18 column (Thermo 72105–254630). mass spectrometry. Cleaved peptide was separated from resin by For confocal imaging experiments lyophilisate was resuspended filtration. Peptide was precipitated with cold diethyl ether; pellet in 50% (vol/vol) ACN and aliquots stored at −80 °C. GxTX-dy550 was washed once with ether and dried under a stream of N2. lyophilisate appeared sparingly soluble in 50% ACN and prone to Peptide pellet was dissolved in water with (by volume) 50% aggregation. To enhance solubility and prevent aggregation in acetonitrile (ACN) or 50% acetic acid (AcOH), injected onto subsequent electrophysiology experiments it was resuspended in a preparatory C18 column (Vydac 218TP101522), and eluted 1 M arginine HCl, 50 mM , pH 5 with NaOH, and with an increasing concentration of ACN with 0.1% TFA. Re- aliquots were stored at −80 °C. covered peptides were lyophilized, dissolved in 50% (vol/vol) GxTX Pra13 was conjugated to methoxy-PEG-Azide with an μ ACN, diluted to 50 M, and folded by air oxidation in 1 M average mass of 5 kDa (Creative PEGWorks PLS-2024) using guanidinium HCl, 0.1 M ammonium acetate, 2.5 mM GSH, 0.25 copper(I)-catalyzed azide-alkyne cycloaddition in the presence of mM GSSG, 1% ACN, pH 8 with ammonia. Oxidation was the catalytic ligand BTTAA (bis[(tertbutyltriazoyl)methyl]-[(2- monitored by mass spectrometry (Applied Biosystems SCIEX carboxymethyltriazoyl)methyl]-amine) (1). Reagents were added TF4800 MALDI TOF-TOF). Upon completion (3 d), the visible sequentially to a polypropylene tube: 5 μL 1 M sodium phos- aggregates that had formed in solution were removed by filtra- phate buffer, pH 7; 24 μL 2.5 mM CuSO4; 15 mM BTTAA; then tion, and 0.1% TFA added. This solution was pumped onto 15 μL 1 mM methoxy-PEG-azide; 15 μL DMSO; 15 μL 1.5 mM a C18 column, eluted as above, and lyophilized. GxTX Pra13; 10 μL 150 mM sodium ascorbate. The reaction mixture was briefly vortexed after each addition. The reaction His6-GxTX Expression. Oligonucleotides encoding the 36 amino was shielded from light, mixed with 1,000 rpm shaking at 25 °C + acid-GxTX sequence were ligated into a pET-30a( ) plasmid for 4 h before quench with 50 μL 10 mM EDTA and HPLC between the N-terminal hexahistidine tag and an enterokinase purification as described above. cleavage site at the C terminus of Venus yellow fluorescent Purity of conjugates was further confirmed by Tris/Tricine SDS/ protein. The His6-GxTX-Venus fusion protein was expressed in PAGE (Fig. S1D). Five hundred ng of each peptide or 0.7 μl × Escherichia coli strain BL21 (DE3). Cells were grown using 2 polypeptide standard (Bio-Rad 161–0326) was diluted in tricine YT culture medium in a shaker incubator at 37 °C until an op- sample buffer (Bio-Rad 161–0739) with 2% (vol/vol) 2-mercap- tical density of 0.5 was reached, and 1 mM isopropyl-1-thio-β-D- toethanol and denatured at 95 °C for 5 min. Samples were loaded galactopyranoside was added to induce expression. After 4 h, into a 10–20% (wt/vol) polyacrylamide gel (Bio-Rad 456–3116) cells were pelleted, resuspended in 20 mL of PBS, and then lysed with 100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3 running by heating to 80 °C for 7 min. The lysate was cooled on ice, and buffer. Gels were run at 30 V until dye entered gel (∼10 min), 1 mM PMSF, 1 mM MgCl2, and 0.1 mg DNaseI were added. then 100 V until dye reached the bottom of the gel (∼45 min). After 20 min, cells were centrifuged at 25,000 × g to remove Gels were fixed in 10% acetic acid, 40% (vol/vol) ethanol for 60 precipitate, and the supernatant concentrated by spin dialysis min, washed twice for 5 min in water, and stained overnight in (Amicon Ultra 30-kDa molecular weight cutoff). The fusion a colloidal Coomassie stain (2) composed of 0.12% Coomassie protein was purified using nickel affinity FPLC, eluting with an G-250, 10% (wt/vol) ammonium sulfate, 17% (wt/vol) o-phos- imidazole gradient. Imidazole was removed by spin dialysis, and phoric acid, and 20% (vol/vol) methanol. Gels were destained in the GxTX domain, as part of the fusion protein, was refolded by water at 4 °C, and imaged digitally (Bio-Rad 170–8270). air oxidation as above. Refolded His6-GxTX was cleaved from Synthesis of the azide beads was initiated by swelling amine Venus using recombinant enterokinase (1 U/50 μg substrate, No- functionalized (0.26 mmol/g) dendrimetic resin beads (Rapp- vagen), then subsequently purified by size exclusion FPLC to obtain polymere S30902) with DMF for 12 h in a polypropylene tube. pure His6-GxTx (Sequence: MHHHHHHSTS EGECGGFWWK Azido-PEG4-NHS (Conju-Probe), 3 equiv, and N,N-diisopropyl-

Tilley et al. www.pnas.org/cgi/content/short/1406876111 1of7 ethylamine (3 equiv), dissolved in DMF, were added to the sus- and digitized at 100 kHz. All recordings were made after addition pended resin beads. The tubes were placed on a rotator for 8 h of 0.1% BSA. Toxins were added by flushing 100 μL through until negative Kaiser tests confirmed complete coupling. The ob- a low-volume recording chamber (Warner R-24N). For dissocia- tained azide-functionalized beads were washed with dichloro- tion rates, the chamber was under constant perfusion of the ex- methane, methanol, and DMF, respectively, three times each, then ternal solution at 2 mL/min and 200 μL of toxin was perfused dried and stored at 4 °C. For conjugation, beads were swollen for through the recording chamber while holding at −100 mV. Toxin 1 h in DMF, rinsed three times each with 1:1 DMF:water, water, binding rate and affinity with a −100 mV holding potential was then 0.5 M sodium phosphate, pH 7. GxTX Pra13 was conjugated measured from the change in current level at the end of 100-ms to beads by copper-mediated azido-alkyne condensation. Azide- steps to 0 mV. These test pulses were repeated every 2 s. Toxin functionalized beads were reacted with alkyne-functionalized binding rate and affinity with a 0-mV holding potential was molecules. To 2-mg beads in 122 μl 0.5 M sodium phosphate, measured by continuous recording at 0 mV after inactivation had pH 7, in a 1.5-mL polypropylene tube, reagents were added and reached steady state. P/N subtraction was not used with these briefly vortexed after each addition: 20 μl of 200 μMGxTXPra13; protocols. Cells were induced longer to have more channels for 8 μl2.5mMCuSO4, 15 mM BTTAA; 30 μlDMSO;10μL 150 these experiments. It is not known whether toxin interacts dif- mM sodium ascorbate. For fluorophore labeled beads, GxTX so- ferently with activated vs. inactivated states. Toxin dissociation lution was substituted with water, and DMSO included 10 mM al- with a −100 mV holding potential was measured from the change kyne-PEG3-5(6)-carboxytetramethylrhodamine (Click Chemistry in current level at the end of 100 ms steps to 0 mV. These test Tools TA108). After 2 h of slow rotation protected from light, pulses were repeated every 10 s. To measure the effect of 0-mV the reaction was quenched with 1 mM EDTA. Beads were rinsed stimulus, 5-s pulses to 0 mV were given every 10 s, with the with water, 50% (vol/vol) DMF, then DMF, slowly rotated for −100-mV interval sufficient to recover from inactivation. 5 min, rinsed with 50% (vol/vol) DMF, three times with water, For experiments with Pra13 GxTX variants, whole-cell patch- and stored in 70% (vol/vol) ethanol at 4 °C until use. clamp experiments were performed with a QPatch-16 automated electrophysiology platform (Sophion Biosciences) using dispos- Cell Culture. CHO-K1 cells were maintained in tissue-culture able 16-channel planar patch chip plates (QPlates; patch hole treated polystyrene dishes (BioLite, Thermo) 37 °C in a 5% CO2 diameter ∼1 μm, resistance 2.00 ± 0.02 MΩ). Intracellular so- atmosphere in Ham’s F-12 media (Corning MT-10–080-CV) lution was same as for manual patch clamp, Qpatch external containing 10% FBS (GemCell 100–500), and 1% penicillin– solution contained (in mM): 10 Hepes, 3.5 KCl, 155 NaCl, – streptomycin solution (Life Technologies 15140 122). Trans- 1 MgCl2, 1.5 CaCl2, adjusted to pH 7.4 with NaOH. Cells were fections were achieved with Lipofectamine LTX (Life Technol- grown to 60–80% confluency in 175-cm2 flasks and harvested by ogies 15338–100) following manufacturer’s instruction, with 2 μL incubating in 2 mL detachin (Genlantis T100100) for 10–15 min Lipofectamine, 1 μg DNA per ml Ham’s F-12 media; media was at 37 °C. Five mL of resuspension solution (in mM: 10 Hepes, – replaced 4 6 h after transfection, and cells used for experiments 2 KCl, 150 NaCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, pH 7.4 with 2 d later. A CHO-K1 cell line expressing rat Kv2.1 (3) was cul- NaOH), was added, then cells were pelleted at 200 × g for 5 min, tured with 1 μg/mL blasticidin, 25 μg/mL zeocin to retain trans- and suspended in 1 mL of resuspension solution. The re- fected vectors. Before experiments, 1 μg/mL tetracycline was suspended cells were then added to the centrifuge of the Qpatch added to media to induce a desirable amount of channel ex- and pelleted at 200 × g for 5 min before commencing automated pression: 1–2 h for most electrophysiology, or overnight for im- electrophysiology protocols. Cell positioning and sealing pa- aging. The BFP cell line was generated from the blue fluorescent rameters were set as follows: positioning pressure −100 mbar, protein variant EBFP2 with a nuclear localization sequence (4), resistance increase for success 750%, minimum seal resistance kind gift of Michael W. Davidson, Florida State University, 0.05 GΩ, holding potential −90 mV, holding pressure −20 mbar. Tallahassee, FL. BFP positive cells were subcloned following Access was obtained with the following sequence: suction pulses selection with 1 mg/mL G418. The cell line was expanded and in 25-mbar increments from −250 mbar to −475 mbar; suction maintained with 100 μg/mL G418, 10 μg/mL blasticidin, and 250 ramp to −450 mbar; 10 × 10 ms −40- mV voltage zaps. Vehicle ug/mL zeocin. The GFP-Kv2.1 expression vector (5) was the kind for toxin application included 0.1% BSA and 5 μM . gift of James Trimmer, University of California, Davis. Rat Electrophysiological current traces shown were digitally smoothed hippocampal neurons were prepared as described (6). with a 1-kHz Gaussian filter for presentation.

Electrophysiology. Whole-cell voltage-clamp recordings were used Live Cell Imaging. Cells were plated in chambered coverglass (Nunc + to measure currents from Kv2.1 channels. Cells were harvested by 155409) and imaged in 5 mM K ringer (in mM: 5 KCl, 135 NaCl, scraping in divalent-free PBS with 1 mM EDTA, pelleted at 1,000 g 2CaCl2,2MgCl2,0.1MgEDTA,50Hepes,20NaOH,pH7.3with for 2 min, resuspended in CHO-SFMII media (Life Technologies HCl) with 0.1% BSA. Cells were incubated in 100 nM GxTX-dy550 12052–114) supplemented with 25 mM Hepes (pH 7.3), and ro- or 120 nM GxTX-TMR. Confocal images were obtained using an tated in a polypropylene tube at room temperature until use. inverted Zeiss LSM510 system with a 1.4 N.A. 63× apochromat oil Aliquots of cell suspension were added to a recording chamber immersion objective. The GFP-Kv2.1 was stimulated using the 488- and rinsed with external solution 5 or more minutes before re- nm line from an argon laser with an HFT 488/543-nm main dichroic cording. The external (bath) solution contained (in mM): 50 beam splitter and BP 505–530-nm emission filter. For GxTX-550 and Hepes, 20 KOH, 155 NaCl, 2 CaCl2,2MgCl2,0.1Mg-EDTA, TMR, a 543-nm helium–neon laser was used with the 488/543-nm adjusted to pH 7.3 with HCl. The internal (pipet) solution con- dichroic and an LP 56-nm emission filter. For time-lapse imaging, tained (in mM): 50 KF, 70 KCl, 35 KOH, 5 EGTA, 50 Hepes, unless otherwise noted, a 530-nm LED light source (Zeiss Colibri) adjusted to pH 7.3 with HCl. A calculated liquid junction potential with 520/28-nm excitation filter, 538-nm dichroic, and 550-nm long- of 6.6 mV was corrected. Pipette tip resistances with these so- pass emission filter set were used. Images were collected with an lutions were less than 3 MΩ. Recordings were at room tempera- EMCCD camera (Photometrics QuantEM 512SC) camera using ture (22–24 °C). Voltage clamp was achieved with an EPC-10 aZeiss40× 0.95 N.A. apochromat air objective, run by the Micro- + amplifier run by Patchmaster software (HEKA). Holding poten- manager software suite 1.4.14 (7). To increase K concentration, + + tial was −100 mV. Series resistance compensation was used when a solution with K replacing Na was added manually. For patch- needed to constrain voltage error to less than 10 mV. Unless clamp fluorometry, the pipet solution contained (in mM): 155 N- otherwise indicated, capacitance and Ohmic leak were subtracted methyl-D-glucamine, 50 HF, 5 EGTA, 50 Hepes, adjusted to pH 7.3 using a P/5 protocol. Recordings were low-pass filtered at 10 kHz with HCl; a tube lens magnification of 2.5× was used and the camera

Tilley et al. www.pnas.org/cgi/content/short/1406876111 2of7 exposure, light source, and patch-clamp recordings were synchro- is the dissociation constant for GxTX, and [GxTX] the concen- nized using the electrophysiology software; in Fig. 4 D and F,anLD- tration of GxTX applied. C apochromat 63×/1.15 water immersion objective and images were Toxin association (kon) and dissociation (koff) rates were de- collected with an EMCCD camera (QImaging Rolera Thunder) termined by camera,runbyZEN2012(Zeiss).Fig.4D–G and Movie S1 were 8 > " obtained using a pipet solution that contained (in mM): 5 KOH, 30 <> CsOH, 70 CsCl, 50 NaF, 50 Hepes, 5 EGTA, adjusted to pH 7.3 IK 1 1 = 1 − + with HCl. In Fig. 4G, trains were approximated by > + + IKmax :> 1 koff k ½GxTX 1 koff k ½GxTX epochs stimulating voltage steps from a holding potential of −80 on ∞ on 0 mV to +40 mV at a frequency of 100 Hz for 50 s. 9 ! #>4 => Bead Assays. GxTX-bead conjugates were rinsed three times with 1 −ð ½ + Þ − kon GxTX∞ koff t ; water, then added to suspended CHO cells. To assess bead binding + e > 1 koff ½ > to cells, CHO-K1 cells or the CHO-K1 cell line expressing Kv2.1 kon GxTX ∞ ; were grown to ∼80% confluency on 14-cm–round tissue culture dishes. Cells were incubated with 1 μg/mL tetracycline for 1 d [S3] before experiments to express Kv2.1. Cells were harvested as de- scribed and each dish resuspended in 2 mL media in a 2-mL where IK/IK max is fraction of current remaining after application polypropylene tube. GxTX-coated and rhodamine control beads of GxTX with respect to time, Kd is the dissociation constant for were added to cell suspensions, mixed by inverting the tube, and GxTX, [GxTX]0 is the initial concentration of GxTX, and [GxTX]∞ is the steady-state concentration of GxTX. For disso- allowed to settle and make adhesive contacts. Tubes were in- = cubated at room temperature and inverted every 15 min to allow ciation, kon[GxTX] 0, and koff values were adjusted for the new bead–cell contacts to form. After 1 h, aliquots of beads were fraction of time channels were stepped to 0 mV. For association, k was constrained to the mean value measured in dissociation removed for visual inspection. Incubations were continued up to off experiments. 3 h, until a majority of GxTX coated beads were bound to Kv2.1 Fluorescent images were analyzed using ImageJ 1.47 software cells. At this point, bead-coated cells were split into different sa- (8). The colocalization Pearson’s coefficient was calculated using line solutions, control (in mM: 5 KCl, 155 NaCl, 2 CaCl ,2MgCl , 2 2 the JACoP plugin (9). Epifluorescent regions of interest (ROIs) 0.1 MgEDTA, 50 Hepes, 20 NaOH, pH 7.3 with HCl + 0.1% were selected manually. Background was defined as the mean BSA) or high [K+] (in mM: 160 KCl, 2 CaCl ,2MgCl,0.1 2 2 fluorescence intensity of a region lacking cells. Reported fluo- MgEDTA, 50 Hepes, 20 KOH, pH 7.3 with HCl + 0.1% BSA), rescence intensity and changes (ΔF) were calculated from and placed on a tube rotator. Following at least 20-min rotation, ROI mean fluorescence intensity minus background. Time de- beads were plated into a multiwell plate for quantitation. Beads pendence of fluorescence intensity was fit with a single expo- were scored as having cells bound if four or more cells could be nential decay: seen adhering to beads. Experimenter was blinded to identity of cell type and saline solution when assessing cell binding. Fluo- ΔF −koff t rescence overlays were obtained with a 550/25ex, 605/70em filter = Fo + Ae ; [S4] F set (Zeiss 43HE). where F corresponds to the maximum fluorescence intensity. Data Analysis. Electrophysiology analysis and graphing were per- F is the remaining fluorescent signal after attenuation from formed with IgorPro software (Wavemetrics), which performs o – voltage activity. The variables A, t,andkoff correspond to nonlinear least-squares fits using a Levenberg Marquardt algo- the amplitude, time, and rate constant, respectively. When rithm. Unless indicated otherwise, means are geometric; error bars indicated, fluorescence intensity was fit with a double-expo- indicate SEs; SDs are reported in text from individual fits; statis- nential decay: tical comparisons are with a two-tailed Mann–Whitney two-sample rank-sum test. Conductance values were determined from current ΔF − − = + − kfastt + − kslowt ; [S5] level at the end of 100-ms voltage steps to the indicated values Fo A Fo fe 1 f e + F normalized by the Nernst potential for K . Conductance data were fit using the fourth power of a Boltzmann distribution function: where f corresponds to the ratio of the dominant amplitude over = + the sum of the two components (f Afast/[Afast Aslow]). Inter- 4 = 1 ; [S1] estingly, at 0 mV, fluorescence decay data over longer intervals GK A ½−ð − Þ = 1 + e V Vsubunit zF RT revealed a second, slow component that was fit well with a dou- ble-exponential decay (Eq. S5; dashed blue line in Fig. 4B). The = ± −1 where GK is Kv2.1 conductance, A is maximum amplitude, Vsubunit fast component k0mVfast 0.20 0.10 s of these double- is the activation midpoint of each of four subunits, z is elementary exponential fits was similar to the monoexponential (Eq. S4) −1 charge, F the Faraday constant, R the ideal gas constant, and T fit to the first 20 s of decay, k0mV= 0.20 ± 0.11 s , n = 5 cells. absolute temperature. The midpoint (Vmid)wasthevalueofV A minor kinetic component was slower, k0 mV slow = 0.021 ± when A reaches half-maximum. 0.003, n = 5 and had an amplitude consistently 26 ± 4% of The Kd of GxTX was determined by k0mVfast. The rate of k0 mV slow was also significantly faster than 0 1 k− (P = 0.008 two-tailed Mann–Whitney two-sample rank- 4 100 mV sum test), indicating that this fluorescence component is also B C IK = B − 1 C ; [S2] voltage sensitive. The presence of a slow fluorescence compo- @1 + A IKmax 1 Kd=½GxTX nent was not predicted by electrophysiology. Its physical origins are unknown, and could potentially result from complexities in GxTX binding to cells or self-quenching of multiple VST–fluo- where IK/IK max is the fraction of current remaining after appli- rophores bound to a single channel. To reduce the number of fit cation of GxTX at the end of a 100-ms voltage step to 0 mV, Kd parameters, we used rates from monoexponential fits (Eq. S4)to

Tilley et al. www.pnas.org/cgi/content/short/1406876111 3of7 the initial fluorescence change to report activation of ion chan- quence [Protein Data Bank (PDB) ID code 2R9R] using the nels with this VST probe. ClustalX software (14); 10,000 models of the voltage-sensing The voltage dependence of kΔF was fit with the following domain were generated from the homology template and the Boltzmann distribution: lowest-scoring model was chosen as the best model. The GxTX NMR structure (PDB ID code 2WH9) was minimized and 1 kΔ = kΔ + A ; [S6] docked to the lowest-scoring Kv2.1 VSD model. The GxTX F Fmin ½−ð − Þ = 1 + e V V1=2 zF RT centroid was placed within 20 Å of the centroid of the Kv2.1 residues I273, F274, L275, T276, E277, and S278 (15). Trans- where kΔF is the rate of change of fluorescence, kΔF min the mini- lational and rotational perturbations were induced by Monte mum value of the function, V the midpoint of the function, and 1/2 Carlo methods and side-chain conformations allowed to relax to the maximum value of the function kΔ = kΔ + A. F max F min energetic minima; 10,000 docking complexes were generated Structural Modeling of the Kv2.1 VSD and GxTX. Homology modeling using Rosetta Dock with an implicit membrane and the lowest of the voltage-sensing domain of rat Kv2.1 channel was performed ΔΔG score model was chosen as the best (16, 17). All structural using the Rosetta-Membrane method (10–13). The Kv2.1 se- modeling images were rendered with the UCSF Chimera soft- quence was aligned with the Kv1.2–Kv2.1 chimera channel se- ware package (18).

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Tilley et al. www.pnas.org/cgi/content/short/1406876111 4of7 Fig. S1. Chemoselective side chains retain tarantula toxins bioactivity and permit ligation. (A) Conductance–voltage relations from GxTX variant with Cys(Acm) insertions at indicated position. Data points are from single cells normalized to maximal amplitude of Eq. S1 fit to vehicle control data from same cell. + + + Lines are fits of Eq. S1. Acm1: Vmid = 76 ± 3 mV, z = 0.8 ± 0.2e ; Acm13: Vmid = 66 ± 2mV,z = 1.4 ± 0.3e ; Acm15: Vmid = 84.2 ± 0.3 mV, z = 0.95 ± 0.02e ; + + + Acm17: Vmid = 84 ± 3 mV, z = 0.7 ± 0.2e ; Acm21: Vmid = 77.5 ± 0.5 mV, z = 1.24 ± 0.04e ; 300 nM Acm27: Vmid = 6 ± 3mV,z = 1.7 ± 0.2e ; 1 uM Acm27: Vmid = + + + 55 ± 3 mV, z = 1.4 ± 0.2e ; 1 uM Acm32: Vmid = 8 ± 2 mV, z = 1.0 ± 0.1e ;3.8μMHis6: Vmid = 31 ± 3 mV, z = 0.7 ± 0.1e . Dotted lines are fits to vehicle and 1 μM GxTX from Fig. 1B.(B) Reaction scheme of azide–PEG conjugation to a propargylglycine residue to form GxTX–PEG conjugate. (C) Reaction scheme of Cys(Acm) deprotection and subsequent tetramethylrhodamine maleimide conjugation to form GxTX conjugate. Proprietary structure of Dylight-550 maleimide could not be shown. (D) Coomassie stained SDS/PAGE gel indicates formation of expected conjugates and lack of contaminants. PEG mobility is less than predicted by molecular weight.

Tilley et al. www.pnas.org/cgi/content/short/1406876111 5of7 Fig. S2. Dylight550 modestly alters GxTX binding to Kv2.1. (A) Normalized dose–response profile of GxTX-dy550 (red circles), −100-mV holding potential. Red line is fit of Eq. S2; where Kd = 30.0 ± 3.9 nM. Black line is fit of Eq. S2 to GxTX dose–response from Fig. 3B.(B) Summary of association rates at −100 mV for GxTX 5 4 -1· −1 −3 −4 −1 and GxTX-dy550. kon = 1.84 × 10 ± 3.03 × 10 M s .(C) Summary of dissociation rates at −100 mV for GxTX and GxTX-dy550. koff = 6.53 × 10 ± 8.33 × 10 s .

Tilley et al. www.pnas.org/cgi/content/short/1406876111 6of7 Movie S1. Complete frame set of fluorescence images used in Fig. 4 D and E. CHO-K1 cells expressing Kv2.1 channels bathed in 10 nM GxTX-dy550. Cell on right is clamped at voltages indicated.

Movie S1

Tilley et al. www.pnas.org/cgi/content/short/1406876111 7of7