Figures & data
Figure 1. AnxA5 inhibits CFTR in Xenopus oocytes. (A) Original traces of the whole-cell current and (B) Summary of whole-cell conductances activated upon stimulation with IBMX and forskolin (I/F, 1mM, 2μM) of oocytes expressing CFTR in the absence (left) or presence (right) of co-expression AnxA5. A reduction in whole-cell conductance was observed when AnxA5 was co-expressed. (C) The inhibitory effect of AnxA5 on whole-cell currents is equally observed in oocytes expressing the CFTR-mutant S737A/S768A. Open bars indicate the conductance before stimulation, i.e., in the absence of I/F. Mean ± SEM, n = number of experiments. Indicates significant activation of whole-cell conductances by I/F (paired t-test). #Indicates significant difference from cells lacking expression of AnxA5 (unpaired t-test).
Figure 2. AnxA5 inhibits CFTR function in Xenopus oocytes independently of the PDZ domain. (A) Summary of whole-cell conductances activated upon stimulation with I/F (1 mM, 2 μM) of oocytes expressing CFTR with and without AnxA5 co-expression. Effects of pre-incubation with cytochalasin D (Cyto D, 10 μM, 2h) on activation of CFTR. (B) Effects of pre-incubation with dynasore (80 μM, 2 h) on activation of CFTR. (C) Summary of whole-cell conductances activated upon stimulation with I/F (1 mM, 2 μM) in oocytes expressing CFTR mutant E1474X (lacking the PDZ-BD) with and without co-expression of AnxA5. (D) Effects of co-expressing the protein S100A8 on whole-cell conductance of oocytes expressing CFTR and AnxA5. Open bars indicate the conductance before stimulation, i.e., in the absence of I/F. Mean ± SEM, n = number of experiments. Indicates significant activation of whole-cell conductances by I/F (paired t-test). #Indicates significant difference from cells lacking expression of AnxA5 (unpaired t-test).
Figure 3. AnxA5 activates CFTR-currents in HEK293 cells. (A) I/F (100 μM; 2 μM) activated whole-cell currents in HEK293 cells expressing wt-CFTR or co-expressing wt-CFTR and AnxA5. Partial removal of Cl- from the bath solution (32 Cl−) inhibits whole-cell currents, indicating activation of CFTR Cl− currents. (B) Summary of the whole-cell conductances activated upon stimulation with I/F. Patch-clamp experiments performed in the fast whole-cell configuration. Open bars indicate the conductance before stimulation, i.e., in the absence of I/F. Mean ± SEM, n = number of experiments. Indicates significant activation of whole-cell conductances by I/F (paired t-test). #Indicates significant difference from cells lacking expression of AnxA5 (unpaired t-test).
Figure 4. Enhancement of CFTR function by AnxA5 and membrane trafficking in mammalian cells. (A) Summary of the whole-cell conductances induced by I/F in HEK293 cells expressing the CFTR membrane retrieval-mutant Y1424A/I1427A in the absence or presence of AnxA5. (B) Effects of co-expression of AnxA5 on Y1424A/I1427A-CFTR in the presence of the dynamin-inhibitor dynasore (40 μM). (C) Summary of the effects of co-expression of AnxA5 on E1474X-CFTR mutant (ΔPDZ-BD). (D) I/F activated whole-cell conductances generated by the double retrieval/ΔPDZ-BD mutant Y1424A/I1427A/E1474X-CFTR in the absence or presence of AnxA5. Open bars indicate the conductance before stimulation, i.e., in the absence of I/F. Mean ± SEM, n = number of experiments. Indicates significant activation of whole-cell conductances by I/F (paired t-test). #Indicates significant difference from cells lacking expression of AnxA5 (unpaired t-test).
Figure 5. CFTR and AnxA5 do not co-localize in vivo. (A) Confocal images of HeLa cells transiently co-transfected with GFP-AnxA5 and either mCherry-flag-wt-CFTR (upper row) or mCherry-flag-F508del-CFTR (lower row). Columns from left to right: mCherry-CFTR; GFP-AnxA5; overlay of mCherry-CFTR and GFP-AnxA5. (B) Confocal images as in A but after treatment with 10 μM ionomycin for 5 min: HeLa cells transiently co-transfected with GFP-AnxA5 alone (upper row); GFP-AnxA5 and mCherry-flag-wt-CFTR (middle row); or GFP-AnxA5 and mCherry-flag-F508del-CFTR (lower row). Columns from left to right: DAPI nuclear staining (in AnxA5 alone cells) or mCherry-CFTR; GFP-AnxA5; overlay of the two previous columns. Scale bar: 10 μm. Cells were observed using a LSM710 Zeiss confocal microscope. Images are representative of at least n = 50 cells from n = 5 independent transfections.
Figure 6. CFTR and AnxA5 do not co-immunoprecipitate. (A) Western blot (WB) of AnxA5 (upper panel) or Hsc70 (a known CFTR interactor as a positive control, lower panel) in total lysates as control (lanes 1,2) or after CFTR co-immunoprecipitation (lanes 3,4) from stable CFTR-expressing BHK cells either non-transfected (lane 2) or transiently transfected (lanes 1,3,4) with AnxA5. In the total lysate controls for WB detection (lanes 1,2) specific bands for both AnxA5 and Hsc70 can be observed at the correct apparent molecular weight. The higher amount of AnxA5 levels detected in lane 2 shows that the transient transfection of AnxA5 was efficient. For samples undergoing co-immunoprecipitation (lanes 3,4) the lysates from cells transiently transfected with AnxA5 were incubated without antibody, ‘beads-only’ (lane 3) or with (lane 4) anti-CFTR primary Ab (IgG) plus the beads to perform the CFTR pull-down. The CFTR-co-immunoprecipitated proteins were then analyzed by WB for AnxA5 (upper panel) or Hsc70 (lower panel). Although AnxA5 was present in the CFTR pull-down (lane 4, upper panel), it was similarly present in the absence of the anti-CFTR Ab (lane 3, upper panel). The lower band, marked ‘*’ corresponds to the IGg light chain. In contrast, Hsc70 was only present in the CFTR immunoprecipitate (lane 4, lower panel), but not in the absence of the anti-CFTR Ab (lane 3, lower panel). In (B) the CFTR-immunoprecipitated material was analyzed by WB for CFTR as the ‘input’ control of the experiment in (A). In (C), the opposite IP experiment of (A) was performed, i.e., AnxA5 was co-immunoprecipitated from AnxA5-transfected, CFTR-expressing BHK cells using AnxA5 Ab and samples were immunoblotted for CFTR. For enhanced sensitivity, the same WB was exposed for longer (left panel) and shorter periods (middle panel). Presence of CFTR in the AnxA5 pull-down can only be observed in the left panel (high exposure time). As a control, a pull-down of CFTR was performed in parallel (right panel) showing that CFTR could be efficiently immunoprecipitated.
Figure 7. siRNA-AnxA5 suppresses whole-cell conductance generated by wt-CFTR: (A) Representative original recording from an open-circuit Ussing chamber experiment with H441 cells grown on permeable supports showing the negative deflection of the transepithelial voltage upon stimulation with I/F (100 μM/2 μM), indicating activation of CFTR-dependent Cl− secretion. Incubation of the cells with siRNA for AnxA5 reduced I/F-induced Cl− secretion when compared to cells treated with scrambled (scrbld) siRNA. (B) Calculated equivalent short-circuit current (Isc) activated by I/F (100 μM/2 μM) in cells treated with scrambled or AnxA5-siRNA. Mean ± SEM, n = number of experiments. #Indicates significant difference from cells treated with scrambled siRNA (unpaired t-test).
