Figure 6. Identification of a PLS domain in ANO6.

(A) Site-specific Type II divergence scores were calculated by comparing ANO1 and ANO6 sequences from 24 mammalian species, binned over a 15-amino acid window, and normalized to the maximum value. Horizontal lines at top indicate transmembrane domains. Vertical lines indicate individual amino acids with high Type II Divergence Scores. (B) Summary of 1-6-1 chimeras. Black line represents ANO1 sequence with TMDs 2–10 labeled. Colored horizontal lines represent ANO1 sequence that was replaced with the corresponding ANO6 sequence. Numbers refer to ANO1 sequence. Green: ANO1-like currents, no scrambling. Red: scrambling. Grey: weak plasma membrane expression, but no currents or scrambling. Orange: some plasma membrane expression, no currents or scrambling. (C) Scrambling domain (SCRD). The sequences of ANO1 (535–601) and ANO6 (506–572) are aligned with TMD 4 and TMD5 indicated. Amino acids are colored according to Rasmol. SCRD shows region associated with PLS. Asterisks show amino acids that are essential for PLS in ANO6. Figure 6—figure supplement 1 lists sequence accession numbers used for Diverge analysis. Figure 6—figure supplement 2 shows alignment of mANO1 and mANO6 used for chimeric construction. Figure 6—figure supplement 3 tabulates the properties of all of the 1-6-1 chimeras. Figure 6—figure supplement 4 shows a confocal image of scrambling by the 1-6-1_(554-588) chimera. Figure 6—figure supplement 5 summarizes properties of mutations in ANO6 SCRD.

DOI: http://dx.doi.org/10.7554/eLife.06901.008

Figure 6—figure supplement 1. Genbank accession numbers of sequences of mammalian species ANO1 and ANO6 used for DIVERGE analysis.

DOI: http://dx.doi.org/10.7554/eLife.06901.009

Figure 6—figure supplement 2. MUSCLE alignment (McWilliam et al., 2013) of mANO1(ac) and mANO6 used for constructing chimeras.

Blue lines indicate membrane helices 1–10 determined by a homology model based on nhTMEM16 (Brunner et al., 2014).

DOI: http://dx.doi.org/10.7554/eLife.06901.010

Figure 6—figure supplement 3. Properties of 1-6-1 chimeras that trafficked to the plasma membrane and generated ionic currents.

Cells were transiently transfected with the indicated constructs in which the indicated amino acids of mANO1(ac) were replaced with the corresponding amino acids of mANO6 (as determined by the alignment shown in Figure 5—figure supplement 2). Currents were measured with 20 µM free Ca²⁺ in the patch pipet at +100 mV or with zero Ca²⁺ in the patch pipet at +200 mV. PLS was measured by Annexin-V-Alexa568 binding to cultures of cells 10 min after addition of 10 µM A23187 and 1 µM thapsigargin. PLS was assessed in >3 independent experiments, counting >100 cells.

DOI: http://dx.doi.org/10.7554/eLife.06901.011

Figure 6—figure supplement 4. Properties of 1-6-1 chimeras in which pairs or triplets of amino acids were mutated.

None of these chimeras exhibited PLS.

DOI: http://dx.doi.org/10.7554/eLife.06901.012

Figure 6—figure supplement 5. Properties of ANO6 with mutations in the SCRD.

Various mutations in the SCRD abolish PLS and ionic currents.

DOI: http://dx.doi.org/10.7554/eLife.06901.013