FIGURE S1: Sequence alignment of the TMEM16 proteins used in this study. Protein sequence alignment of the two human TMEM16E isoforms, mouse ...
Supplementary Figures
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LEGENDS FIGURE S1: Sequence alignment of the TMEM16 proteins used in this study. Protein sequence alignment of the two human TMEM16E isoforms, mouse TMEM16F, mouse TMEM16B and mouse TMEM16A (isoform ac). Predicted trans-membrane domains, based on nhTMEM16 and mTMEM16A protein structures (Brunner et al., 2014; Paulino et al., 2017), are shown in grey. Extracellular and intracellular amino acid residues are highlighted in blue and green, respectively. Residues of the putative scrambling domain in TMEM16E and TMEM16F (Gyobu et al., 2015; Yu et al., 2015) are indicated in bold. The position of the GDD-related T498I exchange in TMEM16E is highlighted in red. Alignment was performed using CLUSTAL OMEGA (1.2.4) (Web Services of EMBL-EBI). FIGURE S2: Vthreshold of TMEM16E currents shifts negative with increasing cytosolic Ca2+ concentrations. Whole-cell patch-clamp recordings in CHO cells transfected with TMEM16E898-EGFP, using pipette solutions containing zero Ca2+ (a), 3 µM free Ca2+ (b) and 240 µM free Ca2+ (c). Upper panel, current traces elicited by voltage steps ranging from -100 to +180 mV with 20mV increments. Lower panel, same recordings shown at smaller amplitude scales, in a voltage range close to the threshold potential of current activation (value in bold).
In order to illustrate the Ca2+ dependence of TMEM16E current activation, recordings with approximately comparable current amplitudes are shown. Note that these recordings are not representative of the mean current amplitudes determined at the respective Ca2+ concentration (Figure 4). FIGURE S3: Time-dependent modification of membrane currents in combined patchclamp/ PLS experiments.
Whole-cell patch-clamp recordings in response to repeated voltage stimulation (-100 to +180 mV with 20-mV increments) at the indicated time points, using a pipette solution containing 3 µM free Ca2+: (a) HEK293 cell transfected with TMEM16E898-EGFP in standard bath solution, (b) HEK293 cell transfected with TMEM16E898-EGFP in bath solution supplemented with Alexa Fluor 555-conjugated annexin-V, (c) non-transfected HEK293 cell in bath solution supplemented with Alexa Fluor 555-conjugated annexin-V.
