Fig 1: Localization of AnxA1 in the ER lumen.(A–B) Confocal images of permeablized A549 (A) and HeLa (B) cells. Native ANXA1 (red), calnexin (green), nuclei (blue). ANXA1 co-localized with calnexin in nuclear envelope (white arrow). ANXA1 also found in puncta without (white arrowhead) or with (cyan arrowhead) calnexin. (C–D) Confocal images of intact (C) and digitonin permeabilized (D) Panc-1 cells. Native ANXA1 (red), nuclei (blue). White arrow in (D) indicates nuclear envelope labeled with ANXA1. (E) ANXA1-(red) and ß-actin-(blue) capture ELISA of supernatant (left) and whole-cell lysate (right). Dots: signals detected in individual assays (n = 6); horizontal bars: mean, s.e.m.Figure 6—source data 1.Abundance of annexin proteins (quantified as total spectral counts by mass spectrometry analysis) detected in experiment eluates collected from magnetic beads covalently linked to peptides with modified pL2 sequence, and in control eluates collected from beads linked to peptide with scrambled modified pL2 sequence.The spectral count entry is black when the count ratio (experiment count/control count) is =1.2; grey when 1.2 > count ratio=1.0; and grey italics when count ratio <1.0. lfsr is the local false sign rate (see Statistical analysis of mass spectrometry output in the Materials and methods section). The lfsr =0.2 (corresponds to a global false discovery rate =5%) are bolded.
Fig 2: L2 peptide, but not scrambled L2 peptide, was able to compete ANXA1 from the InsP3R channel and restore high channel activity.(A–B) Typical current traces in cyto-out experiments with pipette solutions containing 300 Ca2+ER, 1 µM ANXA1, and 40 µM of L2 (A) or scrambled L2 (sL2) (B) peptides. Peptide sequences shown below corresponding traces. (C) Po of individual current traces (orange: L2 peptide; yellow: sL2 peptide) and averages and s.e.m. (horizontal bars) observed under conditions in (A–B). For comparison, averages and s.e.m. of Po in similar cyto-out experiments without peptide, and without (magenta bars) and with (light green bars) ANXA1, from Figures 4J and 6F, respectively.
Fig 3: [Ca2+]ER inhibition of InsP3R-3 channel activity is mediated by a specific interaction between the channel and ANXA1 in the ER lumen.(A–E) Typical current traces of InsP3R-3 channels in cyto-out configuration with 1 µM ANXA1 and various [Ca2+]ER in pipette solution. In C–E with [Ca2+]ER >40 µM, channel conductance reduced due to permeant-ion block. (F) Po of individual current traces (blue symbols) and averages and s.e.m. (red horizontal bars) observed under conditions in (A–E). Red curve: least-squares fit to average Po at various [Ca2+]ER using simple inhibitory Hill equation with parameters tabulated. (G) Averages and s.e.m. of normalized probability of detection of InsP3R channels (Pd) in cyto-out experiments with 0 (black) or 1 µM (red) ANXA1 in pipette solutions with various [Ca2+]ER. (H–I) Averages and s.e.m. of InsP3R-3 channel open (H) and closed (I) durations in cyto-out patch-clamp experiments in (F). (J–K) Typical current traces in on-nuc patches with pipette (cytoplasmic) solutions containing 2 µM Ca2+i and 10 µM InsP3, with 0 (J) or 1 µM (K) ANXA1. Bath solution: 70 nM Ca2+ER with 0-MgATP. (L) Po of individual current traces (brown: no ANXA1; light blue: 1 µM ANXA1 on cytoplasmic side) and averages and s.e.m. (respective horizontal bars) observed under conditions in (J–K).
Fig 4: Modulation of homotetrameric InsP3R channel activity by native inhibitory peripheral luminal protein or ANXA1 under various experimental conditions: 70 nM, 300 or 600 µM Ca2+ER; in the absence or presence of peptides with L2 sequences (pL2, L2 or sL2); and in on-nuc or cyto-out patch-clamp configurations.Averages and s.e.m. of InsP3R-3 Po are shown as tabulated.
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