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1 Supplementary information Supplementary methods FRET efficiency quantification Briefly, two spectral images were collected from each group, using 405 and 514 nm excitation laser lines. The ECFP only expressing cells, excited by the 405 nm laser line, produced emission spectra (F405), which was then normalized and subtracted from the emission spectra of FRET expressing cells to generate a mix of FRET and direct EYFP excitation signals ("bleed-through"). This signal from the FRET expressing cells was further divided by the emission signal generated with the 514 nm laser line (F514) of the same cell and was defined as Ratio A. In order to eliminate the signal contamination, EYFP alone expressing cells were processed through the same procedure and a bleed-through ratiometric index (A0) was calculated. Subtraction of ratio A0 from ratio A (RatioA-RatioA0) is considered as FRET efficiency and provides a genuine FRET signal. Note that ratio A0 must be less than 0.1 in order to maintain linearity. Voltage-dependent FRET quantification For this purpose, a two-electrode voltage clamp setup was mounted on the confocal microscope (Zeiss LSM 510 META). Electrophysiological recording was performed as previously described1. Briefly, voltage-clamp measurements were performed at room temperature (22oC-24oC) 2-5 days following DNA microinjection. Oocytes were placed into a glass bottom 35mm dish (Mat-Tek Corporation) under low rate perfusion of modified ND96 solution (containing 0.1 mM CaCl2). Whole-cell currents were recorded using a GeneClamp 500 amplifier (Molecular Devices). Stimulation of the preparation, and data acquisition were performed using the pCLAMP 8.1 software (Molecular Devices) and a personal computer interfaced with a Digidata 1322 interface (Molecular Devices). Glass 2 microelectrodes (A-M systems, Inc) were filled with 3M KCl and had tip resistances of 0.20.5 MΩ. Current signals were digitized at 1 kHz and low pass filtered at 0.2 kHz. The voltage clamp protocol included 60 seconds at -80 mV (holding potential), voltage step to +30 mV for a period of 60 seconds and return to -80 mV for another 90 seconds (altogether 210 seconds). Concomitantly with the current recording, 42 spectral images (449-599nm) were acquired at 5 seconds intervals (0.2Hz) under 405 nm laser excitation. FRET ratio was obtained by division of the peak EYFP spectral region signal F[524-534] with the peak ECFP spectral region signal F[481-492] for every point along the time series. In order to calculate the FRET activation ratio, we first normalized the signal to a mono-exponential fit corresponding to the photobleaching occurring during the experiment2 (Supplementary Fig. 6). Then, the Bleach-corrected normalized voltage-dependent FRET F[524-534]/F[481-492] change was deduced by the FRET ratio obtained at the end of the +30mV depolarizing pulse (120 sec) divided by the FRET ratio obtained at -80 mV before the step depolarization (50 sec). Characterization of the Kv7.1 C-terminus/CaM/KCNE1 C-terminus ternary complex E. coli BL-21 Tuner (Novagen), containing the "RIL" Codon PlusTM plasmid (Stratagene) were co-transformed with plasmids pETDuet-1 and CDFDuet-1and grown at 37° C in LB medium supplemented with 100 µg/ml ampicillin, 25 µg/ml streptomycin and 34 µg/ml chloramphenicol. Upon reaching an OD600 of 0.3, the temperature was lowered to 16° C, and the growth was continued until the culture reached an OD600 of 0.6. Protein expression was induced with 135 µM isopropyl 1-thio-β-D-galactopyranoside. Cells were harvested after 14 h by centrifugation, frozen, and suspended in lysis buffer (buffer L: 150 mM NaCl, 50 mM sodium phosphate, pH 8, 1 mM phenylmethylsulfonyl fluoride). Lysis was performed by microfluidizer (Microfluidics), followed by centrifugation at 20,000 x g. The soluble fraction 3 was loaded onto a pre-equilibrated metal chelate Ni2+-NTA (Qiagen) column (buffer A: 300 mM NaCl, 50 mM sodium phosphate, pH 8) at a flow rate of 1.5 ml/min. The column was washed with buffer A, containing 10 mM imidazole, until a stable baseline was achieved. The proteins were eluted with buffer A, supplemented with 250 mM imidazole and the eluate was loaded onto a pre-equilibrated desalting (GE Healthcare) column (buffer A). The elution peak was then subjected to TEV protease at a ratio of 1:50 (weight:weight) for 14 h. The sample was loaded again onto pre- equilibrated metal chelate Ni2+-NTA column (buffer A). The proteins were eluted with buffer A, supplemented with 10 mM imidazole. Fractions were pooled and applied to a pre-equilibrated analytical Superdex 200 gel filtration column (GE Healthcare) with buffer F (200 mM NaCl, 20 mM Tris, pH 7.5, 1 mM dithiothreitol). The elution peak fractions were incubated at -20° C with trichloroacetic acid, followed by centrifugation at 18,000 x g. The supernatant was removed and the pellet was resuspended with sample buffer, and analyzed by SDS-PAGE using a Tris-Tricine gel. Supplementary References 1. Gibor, G., Yakubovich, D., Peretz, A. & Attali, B. External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites. J Gen Physiol 124, 83-102 (2004). 2. Gibson, S.K. & Gilman, A.G. Gialpha and Gbeta subunits both define selectivity of G protein activation by alpha2-adrenergic receptors. Proc Natl Acad Sci U S A 103, 212-7 (2006). 4 Supplementary Figure legends Supplementary Figure 1. Image analysis and FRET signal calculation: (A) raw data obtained from a typical experiment. 405nm diode 30mW laser was used for ECFP/FRET acquisition (with spectral window of 443-599nm), 514nm Argon 30mW laser was used for EYFP/FRET acquisition (with spectral window of 518-599nm due to limitation of the HFT405/514/633 beam splitter). (B) Correction of mean normalized ECFP spectrum (pink – correction of the blue to the green spectrum in (A)) with the FRET spectrum enables subtraction (shown in red). This FRET signal is contaminated with direct EYFP emission (F405) due to small bleed-through. (C) Demonstration of bleed-through component interfering with FRET signals (yellow lines). To eliminate these interferences, a ratiometric approach was used: the emissions from FRET and EYFP only oocytes were divided by the emissions generated with the 514nm laser (which excites only EYFP). (D) RatioA0 represents direct excitation of EYFP with 405nm laser in EYFP-only oocytes, and Ratio A is the same ratio in the FRET oocytes (generated both by FRET and direct EYFP contamination). Subtraction of these ratios yields a pure FRET signal (RatioA-RatioA0) (not shown). Supplementary Figure 2. Electrophysiological properties of fluorescently-labeled subunits. (A) Representative traces of K+ currents measured from Kv7.1(WT) and the fluorescently-labeled constructs expressed in Xenopus oocytes. From a holding potential of – 80 mV, cells were stepped for 3 sec from –70 or -60 mV to +40 mV in 10 mV increments and then repolarized for 1 sec at –120 mV tail potential. (B) I/Imax-voltage relations of WT Kv7.1 and fluorescently-tagged subunits. (C) I/Imax-voltage relations of WT Kv7.1 coexpressed with WT KCNE1 or with KCNE1-EYFP. (D) I/Imax-voltage relations of fluorescently-tagged Kv7.1 and KCNE1 subunits (n = 6-8 cells). 5 Supplementary Figure 3. The non-dimerizing A206K mutants of EYFP or ECFP does not interfere with FRET measurements. The FRET efficiencies, expressed as the mean value of [RatioA−RatioA0] and SEM are shown for the expression of 1:1 molar ratio of Kv7.1-ECFP/Kv7.1-EYFP, Kv7.1-ECFP/KCNE1-EYFP and Kv7.1622-ECFP/KCNE1EYFP, expressed as WT (blank bars) or as A206K non-dimerizing EYFP/ECFP mutant (black bars). Asterisks indicate significance level (* p < 0.05). (n = 15-26 cells). Supplementary Figure 4. Co-expression of CTD hinders Kv7.1 from reaching the plasma membrane. Confocal immunofluorescence images of non-permeabilzed CHO cells, transfected with 2xHA externally tagged-Kv7.1 in the absence (upper panel) or presence of CTD (lower panel). The 2xHA tag was engineered at the external S3-S4 linker, after amino acid 218. Supplementary Figure 5. Electrophysiological properties of the Kv7.1 mutant 386-504. (A) Representative traces of K+ currents measured in CHO cells transfected with WT Kv7.1 (left panel) or mutant 386-504 lacking the linker connecting helices A and B (right panel). From a holding potential of –90 mV, cells were stepped for 2 sec from –70 mV to +60 mV in 10 mV increments and then repolarized for 1 sec at –60 mV tail potential. (B) Representative traces of K+ currents measured in CHO cells co-transfected with WT KCNE1 and WT Kv7.1 (left panel) or mutant 386-504 (right panel). Currents were recorded as in a. (C) Conductance-voltage relations of current recorded from WT Kv7.1 (black squares) or mutant 386-504 (black triangles). Curves were fitted to one Boltzmann function. The following values were obtained: V50 = -40.1 ± 4.5 mV, s = 20.2 ± 2.1 mV (WT Kv7.1); V50 = -39.0 ± 1.2 mV, s = 11.5 ± 1.0 mV (n = 6-8 ) (386-504). (D) Conductance-voltage relations of current recorded from WT Kv7.1 (black squares) or mutant 386-504 (black triangles) in the 6 presence of KCNE1. Curves were fitted to one Boltzmann function. The following values were obtained: V50 = +3.8 ± 1.0 mV, s = 17.6 ± 1.0 mV (WT Kv7.1+KCNE1); V50 = +7.2 ± 1.0 mV, s = 18.0 ± 1.0 mV (n = 9-11) (386-504+KCNE1). Supplementary Figure 6. SDS-PAGE gel profile of purified Kv7.1 and KCNE1 Cterminal peptides. Commassie stain of His-tagged Kv7.1 C-terminal constructs co-purified with CaM, (A) and of purified GST-KCNE1 C-terminal polypeptides (B). Supplementary Figure 7. Voltage-dependent FRET bleach correction. Subtraction of photobleaching was essential in order to obtain quantitative data of the dynamic FRET experiments. (A) Example of a photobleaching fit correction of averaged (n = 14) FRET ratio of EYFP-Kv7.1-ECFP. The dotted line represents a single exponential fit to points 1-12 (0-60 sec), used to approximate the photobleaching decay during the time measurement. (B) Photobleach-corrected time course produced by subtravting the exponential decay from the data in (A).