Supplementary Figure S1. MscS orientation in spheroplasts and liposomes (a) Current-voltage relationship for wild-type MscS expressed in E.

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1 a b c Supplementary Figure S1. MscS orientation in spheroplasts and liposomes (a) Current-voltage relationship for wild-type MscS expressed in E. coli giant spheroplasts (MJF465) and reconstituted into 100 % azolectin liposomes. (b) Representative current traces of MscS reconstituted into 100 % azolectin liposomes recorded at + 30 mv pipette potential in the absence of trifluoroethanol (TFE) (left), application of 2.5% vol TFE to the bath (middle) and in the presence of 2.5 % vol TFE in the patch pipette (right), respectively. (c) Time course of normalized maximum current of MscS reconstituted into 100 % azolectin liposomes in the absence of TFE ( ), application of 2.5% vol TFE to the bath (cytoplasmic side) ( ) and in the presence of 2.5% vol TFE in the patch pipette and applied 2.5% vol TFE to the bath after 30 min ( ), respectively (mean ± SEM; n = 6-10). (a) was adapted and modified from Nomura et al., 2012.

2 Supplementary Figure S2. Channel activity of wild-type MscS and E187R, E227A mutants in the presence of symmetrical 100 BaCl 2 at +70 mv pipette potential (a) There is no difference between subconducting states in any of the three channels. The right panel shows the Coulombic charge map of the vestibular portals of the corresponding channel as viewed from inside the cytoplasmic domain. (b) Table illustrating fully open channel amplitude, average number of subconducting states at +70 mv pipette potential and pressure threshold of activation for WT-MscS, E187R and E227A mutant channels. (c) Table illustrating mean percentage of unitary conductance of each subconducting state at two different voltages (+70 & 90 mv pipette potential) ± S.D. [FO - Fully open, C Closed].

3 Supplementary Figure S3. Comparison of the putative pore-forming residues of six electrophysiologically characterised MscS homologues. (a) MscS TM3 pore-forming residues aligned with putative pore-forming residues of six electrophysiologically characterised MscS homologues with channels arranged with the most anion selective (MSC1) at the top. (b) Histogram illustrating % conservation of consensus residues at each position, the consensus sequence is contained within the histogram. (c) Histogram demonstrating the percentage of hydrophobic residues of the putative pore-forming residues for each homologue. (d) Table illustrating percentage identity (blue values read vertically) and percentage similarity (red values read horizontally) from pair-wise alignments (EMBOSS Needle, Needleman-Wunsch alignment; EMBL-EBI) of putative pore-forming residues. (e) Structure of MscS TM3 poreforming helix (PDB:2VV5) highlighting important residues for gating, inactivation and desensitisation.

4 Supplementary Figure S4. Comparison with MscS of six electrophysiologically characterised homologues Hydrophobicity plots of MSC1 (C. reinhardtii), MscS (E. coli), Sp7 (R. pomeroyi), MscK (E. coli), MscCG (C. glutamicum) MscMJLR (M. jannaschii) and MscMJ (M. jannaschii) arranged with the most anion selective (MSC1) at the top and most cation selective at the bottom (MscMJ)(Kyte-Doolittle scale). Grey shaded area illustrates the conserved cytoplasmic domain of all seven homologues. In the case of MscCG the cytoplasmic domain extends outside the shaded area. Putative transmembrane regions are also shown for comparison. Upper inset shows percentage identity (blue values read vertically) and percentage similarity (red values read horizontally) from pair-wise alignments (EMBOSS Needle, Needleman-Wunsch alignment; EMBL-EBI) of full length proteins. Lower inset shows percentage identity and percentage similarity from pair-wise alignments (EMBOSS Needle, Needleman-Wunsch alignment algorithm;embl- EBI) of putative cytoplasmic domains subsequent to alignment using ClustalW.

5 Supplementary Table S1. Reported relative permeabilities of MscS and corresponding experimental conditions. E rev pipette P Cl /P K Bath Pipette MgCl 2 / ph System Ref. potential solution solution CaCl 2 (mv) () KCl 300 KCl KCl 300 KCl KCl 400 KCl KCl 600 KCl KCl 200 KCl KCl 600 KCl / 10 6 Spheroplasts Sotomayor et al., / n/a 6 Liposomes Sukharev / Spheroplasts Martinac et al., / Spheroplasts Edwards et al., / n/a 7.4 Liposomes This study 90 /10 6 Spheroplasts Li et al., 2002 Supplementary Table S2. Mean percentage of unitary conductance of each MscS subconducting state in symmetrical 100 CaCl 2 at four different pipette voltages ( mv) ± S.D. % Unitary conductance 80 mv 90 mv 100 mv 110 mv SC7 SC6 SC5 SC4 SC3 SC2 SC ±3.9 (n=3) ±0.7 (n=3) ±3.3 (n=3) ±3.0 (n=3) ±5.2 (n=2) n/a (n=1) n/a (n=1) ±2.1 (n=3) ±4.2 (n=3) ±2.4 (n=3) ±1.7 (n=3) ±0.6 (n=3) ±1.1 (n=3) ±1.0 (n=3) ±2.7 (n=3) ±1.6 (n=3) ±1.5 (n=3) ±1.0 (n=3) ±2.2 (n=3) ±2.0 (n=3) ±2.1 (n=3) ±0.4 (n=3) ±2.5 (n=3) ±1.5 (n=3) ±1.2 (n=3) ±4.9 (n=2) ±3.8 (n=3) ±3.1 (n=3)

6 Supplementary Table S3. MscS exhibits higher anion selectivity in BaCl 2 compared to KCl. The table shows permeability ratios (P Cl /P K & P Cl /P Ba ) for WT-MscS, E187R and E227A mutants as calculated in asymmetric KCl (600 pipette/200 bath) and asymmetric BaCl 2 (2.00 pipette/50 bath). Supplementary Table S4. Nucleotide sequence of forward primers used for MscS mutagenesis.