Figure 5. Slc6a14 expression mediates arginine-dependent enhancement of mutant F508del CFTR channel function in murine intestinal tissues.

(a) Hypothetical model depicting that SLC6A14 could affect CFTR channel function. (b) Diagram depicts the concept of gaining apical access to the epithelium by splitting open a 3D organoid, thereby resulting in patches of split-open 2D lawns, which can then be studied using fluorescence-based assays. (c) Split-open colonic organoids from CF (Cftr^{F508del/F508del}) and double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)) mice were studied for CFTR channel function using the previously described membrane potential-based ACC assay. Line graph represents change in fluorescence relative to baseline (ΔF/F₀) as a measure of F508del-CFTR function after low temperature rescue (27°C) of the mutant protein. After capturing baseline fluorescence reads, cells were acutely treated with L-arginine (1 mM) to activate SLC6A14 or vehicle, followed by CFTR activation with cAMP agonist forskolin (FSK 10 µM) or vehicle DMSO. Thereafter, CFTRinh-172 (10 µM) was added to all the wells. (d) Bar graph represents maximum change in ACC fluorescence from baseline (ΔF/F₀) after acute addition of FSK, following low temperature (27°C) rescue of F508del-CFTR protein in split-open murine organoids (mean ± SEM). Paired t-test was performed (*p=0.045, ns = not significant, n = 4 mice for each genotype). (e) ACC assay performed on split-open colonic organoids from CF (Cftr^{F508del/F508del}) and double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)) mice for CFTR channel function at physiological temperature (37°C). As above, SLC6A14 was activated with L-arginine (1 mM) or vehicle followed by CFTR stimulation by FSK (10 µM) or vehicle DMSO. All wells received CFTRinh-172 (10 µM) after activation to confirm the role for CFTR. (f) Bar graph represents maximum change in ACC fluorescence from baseline (ΔF/F₀) after acute addition of FSK, at physiological temperature (37°C) in F508del-CFTR split-open murine organoids (mean ± SEM). Paired t-test was performed (****p<0.0001, ns = not significant, n = 3 mice for each genotype).

Figure 5—figure supplement 1. Slc6a14 is a major arginine transporter in the colonic epithelium.

Ex vivo intestinal closed loop assay was performed in mice on FVB background across four genotypes: Wt, CF (Cftr^{F508del/F508del}), Slc6a14^(-/y) and double mutant (Cftr^{F508del/F508del}; Slc6a14^(-/y)). Each loop for each mouse was injected with buffer containing ³[H]-arginine supplemented with 100 µM or 20 mM cold arginine. Bar graph represents arginine uptake by the epithelium (counts per minute – CPM), normalized to total protein in the lysate (CPM/[protein]), mean ± SD. Two-way ANOVA with Tukey’s multiple comparison test was performed for colon and ileum (**p=0.0013, ***p=0.0007, ****p<0.0001, ns = not significant, n ≥ 4 for each genotype of mouse).

Figure 5—figure supplement 2. Split-open organoid model.

Confocal immunofluorescence images of apical epithelial marker ZO-1 (red) and nuclei (blue) in murine 3D colonic organoid (left panel) and 2D split-open colonic organoids (right panel, shown as XY, YZ and YZ stacked images). Scale bar = 5 µm.

Figure 5—figure supplement 3. SLC6A14 does not enhance processing or cell surface stability of F508del-CFTR in BHK over-expression system.

(a) BHK cells over-expressing mutant F508del-CFTR were transiently transfected with SLC6A14-FLAG or Empty Vector (EV). Cells were lysed and subjected to western blotting. Representative western blots probed with anti-CFTR, anti-FLAG, anti-calnexin, or anti-actin antibodies are shown. (b) Bar graph represents mature CFTR expression calculated as a ratio of CFTR Band C over total F508del-CFTR expression (mean ± SEM). Paired t-test was performed (for EV control transfected p=0.2470, for SLC6A14 transfected p=0.2874, ns = not significant, n = 4 for each group). (c) Cycloheximide Brefeldin A chase was performed on BHK cells over-expressing F508del-CFTR and transiently transfected with EV control or SLC6A14-FLAG. Cells were incubated at 27°C for 24 hr to rescue mutant F508del-CFTR, and then moved to 37°C at the start of the chase experiment. Representative western blots probed for loading control calnexin for CFTR and actin for SL6A14, are shown. (d) Line graph represents decay over time of mature CFTR (Band C) normalized to loading control calnexin in BHK cells transiently transfected with either EV control or SLC6A14. Change in Band C/Calnexin protein expression was normalized to the first time point (t = 0). Two-way ANOVA with Sidak’s multiple comparison test was performed (p>0.05 for all time points, n = 3 biological replicates). (e) Line graph represents decay over time of immature CFTR (Band B) normalized to loading control calnexin in BHK cells transiently transfected with either EV control or SLC6A14. Change in Band C/Calnexin protein expression was normalized to the first time point (t = 0). Two-way ANOVA with Sidak’s multiple comparison test was performed (p>0.05 for all time points, n = 3 biological replicates).

Figure 5—figure supplement 4. SLC6A14 interaction with mutant F508del-CFTR.

BHK cells over-expressing mutant F508del-CFTR and either SLC6A14-FLAG (+) or EV control (-) were used for co-immunoprecipitation. IP = Immunoprecipitation, WB = Western Blot. (a) SLC6A14 was immunoprecipitated with FLAG antibody. Representative western blot of the input lysate before the IP and after the IP are shown. (b) CFTR was immunoprecipitated using the anti-CFTR 596 antibody. Representative western blot of the input lysate before the IP and after the IP are shown. (c) SLC6A14 was immunoprecipitated with FLAG antibody. Representative western blot of the input lysate before the co-IP and after the co-IP are shown. (d) CFTR was immunoprecipitated using the anti-CFTR 596 antibody. Representative western blot of the input lysate before the IP and after the IP are shown.