15 June 2011 Supplementary material Bagriantsev et al.

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1 Supplementary Figure S1 Characterization of K 2P 2.1 (TREK-1) GOF mutants A, Distribution of the positions of mutated nucleotides, represented by a red x, from a pool of 18 unselected K 2P 2.1 (KCNK2) clones following random mutagenesis. Pore-forming regions M1-M4, P1, and P2 are indicated. B, Potassium selectivity of K 2P 2.1 (TREK-1) and GOF channels recorded in Xenopus oocytes in K + /N-methyl-D-glucamine solutions (98.0 mm total) at ph o 7.4. Data represent mean ± SEM (n= 6-7). Error bars are smaller than the symbols. Dashed line represents theoretical Nernst equation E rev =RT/F*log([K + ] o /[K + ] i ), where R and F have their regular physical values, and T=23 o C, assuming [K + ] i =108.6 mm (Guizouarn et al., 2001). C, Quantification of number of active channels per patch from single-channel recordings in COS7 cells expressing K 2P 2.1 (TREK-1) and GOF channels (cell-attached mode, mm K + o, ph o 7.2). Data represent mean ± SE (n = 5-9). D, Current-voltage plots and E, quantification of conductance from single channel analyses of K 2P 2.1 (TREK-1) and GOF channels. Data represent mean ± SEM (n = 4-7).

2 Supplementary Figure S2 Characterization of K 2P 2.1 (TREK-1) GOF mutants A, Immunoblot analysis of total COS7 cell surface expression of K 2P 2.1 (TREK-1) bearing an N-terminal HA tag (HA-K 2P 2.1). Cells were labeled with membrane-impermeable biotinylating reagent followed by cell lysis and capture of the labeled proteins on neutravidin beads. The intracellular GFP expressed from the same vector. B, Quantification of surface expression of K 2P 2.1 (TREK-1) bearing an HA tag in the P1 extracellular loop (K 2P 2.1-HA P1 ). 0.3 ng RNA was injected into Xenopus oocytes for each construct. K 2P 2.1-HA P1 was detected by immuno-labeling of the surface proteins with anti-ha antibody followed by detection with horseradish peroxidase conjugated secondary antibody and a chemiluminescent substrate. Data represent mean ± SE (n = 6-8). TREK1-82HA W275S did not produce a functional channel. C, Exemplar current-voltage traces from whole-cell recordings of Xenopus oocytes expressing constructs from B. Currents were elicited in solutions containing 2 mm potassium (ND96) by a ramp protocol from -150 to +50 mv from a -80 mv holding potential.

3 Supplementary Figure S3 K 2P 2.1 (TREK-1) GOF affect response to extracellular acidosis, temperature, and pressure A, Normalized ph O responses (at 0 mv) for the indicated channels. B, Normalized temperature responses (at 0 mv) for the indicated channels. C, Normalized temperature responses for the indicated channels. For full description, see Figure 2 legend (n = 8-30). N=2-7.

4 Supplementary Figure S4 K 2P 2.1 (TREK-1) GOF mutants are inhibited by external [Mg 2+ ] A-F, Exemplar current-voltage responses of whole-cell recordings of Xenopus oocytes injected with different amounts of K 2P 2.1 (TREK-1) mrna to give comparable current amplitudes. Currents were recorded in the presence of 0 mm or 2 mm [Mg 2+ ] O in 90mM KCl ph 7.4 using 1 s steps from -150 to +50 mv (in +20 mv increments) from a holding potential of 0 mv. Values show measured current after 800 ms. G, Quantification of inhibition by 2 mm Mg 2+ measured at -100 mv. Data represent mean ± SEM (n= 4-5).

5 Supplementary Figure S5 K 2P 2.1 GOF mutants affect gating A and B, Normalized response to ph O in low (2 mm, data is from Figure 2B) and high (90 mm) [K + ] o (2K and 90K, respectively) for K 2P 2.1 (TREK-1) and the indicated GOF mutants. Whole cell currents were elicited in Xenopus oocytes by a ramp from -150 to +50 mv from a holding potential of -80 mv (2K) or 0 mv (90K). C, Quantification of the effect of high (90 mm) external potassium on the pho response from the curves in A and B combined with the data from Figure 3 (n = 6-30). N = 2-7.

6 Supplementary Figure S6 Acidic pho alters K 2P 2.1(TREK-1) ion selectivity Exemplar two-electrode voltage clamp recordings of Xenopus oocytes expressing comparable current amplitudes of K 2P 2.1 (TREK-1) at ph O 9.0 and ph O 6.9 pho, respectively, in 100 mm sodium solution. Currents were evoked by 60 ms long pulses from -150 to -50 mv in 10 mv increments from a -80 mv holding potential.

7 Supplementary Figure S7 Sequence alignment of K 2P channels Putative transmembrane segments are indicated in light blue based on THMM analysis ( of the K 2P 2.1 (TREK-1) sequence. Pore-forming regions, P1 and P2, are indicated in purple. Sites of K 2P 2.1 (TREK-1) GOF mutants are marked.

8 Supplementary Figure S8 Importance of the Trp275 position is functionally conserved across the K 2P channels. A-D, K 2P channel current amplitude at 0 mv in Xenopus oocytes injected with the indicated mrna amounts. Following 24 hrs of expression, currents were elicited by a ramp from -150 to +50 mv from a holding potential of -80 mv, in 2 mm [K+] O, ph 7.4. Data represent mean values at 0 mv ± SEM (n is indicated).

9 Supplementary Table S1 Channel pk 1/2 I min /I ph9.0 n K 2P 2.1 (TREK-1) Wild-type 7.51 ± ± I148T 7.29 ± ± L267P 7.28 ± ± I148T/L267P 7.00 ± ± F276L 7.40 ± ± W275S 7.02 ± ± W275F 7.74 ± ± W275Y 7.61 ± ± W275A 6.42 ± ± W275T 7.35 ± ± W275L 7.17 ± ± W275Q 6.97 ± ± K 2P 10.1 (TREK-2) Wild-type 7.69 ± ± 0.04* 9 W301S 7.96 ± ± 0.01* 9 K 2P 9.1 (TASK-3) Wild-type 6.29 ± ± F225S 7.46 ± ± K 2P 3.1 (TASK-1) Wild-type 7.30 ± ± F225S 8.82 ± ± K 2P 5.1 (TASK-2) Wild-type 8.41 ± ± E228W 6.68 ± ± Supplementary Table S1 [H + ] o gating parameters from whole-cell recordings of K 2P channels and mutants Recorded by two-electrode voltage-clamp of Xenopus oocytes in 2.0 mm K + o at different ph o. Data are mean ± SEM. K 1/2, I min and I ph9.0 were calculated by fitting the data with the Hill equation (I=I min +(I max -I min )/(1+([H + ] o /K 1/2 ) H ). pk 1/2 = logk 1/2 *I max /I ph9.0

10 Supplementary Methods Cell Surface Biotinylation COS7 cells expressing K 2P 2.1 (TREK-1) bearing an HA tag on the N terminus (HA-K 2P 2.1), from the IRES-GFP vector were labeled on ice with the membrane-impermeable Sulfo-NHS-SS-Biotin (Thermo Scientific) reagent accordingly to the manufacturer s instructions. Cells were lysed in a buffer containing 50 mm HEPES ph 7.4, 150 mm NaCl, 1% Triton X100 and antiproteases. Total lysate protein concentration was measured by Bradford assay (Bio-Rad). Biotinylated proteins were precipitated with neutravidin beads from lysates containing 500 ug of total protein, washed, eluted, resolved in denaturing SDS-PAGE and visualized by Immuno-blot using anti-ha (HA-7, Sigma) and anti-gfp (A11122, Invitrogen) antibodies. Cell Surface Chemiluminescence The chemilumenscence assay was done according to (Zerangue et al., 1999) and the following Xenopus oocytes expressing TREK-1 with an HA tag (SGYPYDVPDYASG) inserted after Ile82 in the P1 loop (K 2P 2.1-HA P1 ) for 48 hours were incubated in 24-well plates in cold ND96 solution containing 1% bovine serum albumin (ND96/BSA, 2 ml/well) for 30 minutes on ice, followed by a 30 minute incubation with anti-ha antibody (HA7 from Sigma). Oocytes were washed in 5 wells with cold ND96/BSA to remove excess of the primary antibody, and incubated with horseradish peroxidase (HRP) conjugated secondary antibody (ImmunoPure goat anti-mouse IgG, HRP conjugated, from Thermo Scientific) for 30 minutes on ice. Following an extensive wash (5 wells with ND96/BSA followed by 7 wells with ND96), each oocyte was placed in an eppendorf tube containing 50 µl of the chemiluminescent substrate (Super Signal ELISA Femto from Thermo Scientific). Chemiluminescence was measured immediately for 30 seconds, using the 20/20 Luminometer (Turner Biosystems). References Guizouarn, H., Gabillat, N., Motais, R., and Borgese, F. (2001). Multiple transport functions of a red blood cell anion exchanger, tae1: its role in cell volume regulation. J Physiol 535, Zerangue, N., Schwappach, B., Jan, Y.N., and Jan, L.Y. (1999). A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22,