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. 2017 Jun 1;312(6):L912-L925.
doi: 10.1152/ajplung.00178.2016. Epub 2017 Mar 30.

The CFTR Trafficking Mutation F508del Inhibits the Constitutive Activity of SLC26A9

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Free PMC article

The CFTR Trafficking Mutation F508del Inhibits the Constitutive Activity of SLC26A9

Carol A Bertrand et al. Am J Physiol Lung Cell Mol Physiol. .
Free PMC article

Abstract

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

Keywords: CAL; NHERF-1; PDZ domain; chloride channel; intracellular trafficking.

Figures

Fig. 1.
Fig. 1.
Human bronchial epithelia (HBE) from cystic fibrosis (CF) donors lack a GlyH-101-inhibitable constitutive current as well as a forskolin-stimulated current, suggesting a functional absence of both CFTR and SLC26A9 at the apical membrane. HBE from non-CF donors exhibit both constitutive and forskolin-stimulated currents. Constitutive anion secretion is measured as the short-circuit current (Isc) remaining after inhibition of ENaC with 10 µM amiloride and before addition of forskolin. GlyH-101 (50 µM) inhibits SLC26A9 activity (6), whereas 10 µM forskolin stimulates CFTR activity through activation of the cAMP/PKA cascade. Because GlyH-101 also inhibits CFTR, paired filters were treated with forskolin in the absence of GlyH-101 (Vehicle, dashed lines) to measure CFTR activity. Bumetanide (50 µM) inhibits the basolateral Na-K-2Cl cotransporter. Representative traces from a typical experiment are shown: blue traces, non-CF HBE; and red traces, CF HBE (F508del/F508del). Experiments used a chloride gradient (apical low); the small deflections at 90-s intervals reflect transepithelial resistance (TER) measurements.
Fig. 2.
Fig. 2.
Coexpressing SLC26A9 with F508del CFTR in HEK293 cells significantly reduces constitutive current, compared with coexpression with either wt CFTR or the functionally impaired G551D CFTR. Whole cell patch-clamp measurement of membrane current (Im) or I/V ratio [RIV (6)], observed in HEK293 cells transiently transfected with myc-SLC26A9 and the indicated CFTR construct (left), or either myc-SLC26A9 or wt CFTR alone (right). A: constitutive current was measured 1 min after break-in and before any treatments. B: after measurement of constitutive Im, cells were treated with 10 µM forskolin and the change in ImIm) recorded when the response plateaued (~2 min after forskolin addition). C: after measurement of the forskolin-stimulated ΔIm, cells were treated with 50 µM GlyH-101 (in the continued presence of forskolin), and the RIV was measured to confirm the identity of the conducting channel(s). myc-SLC26A9 exhibits mild rectification (RIV ≅ 0.8) when it is the primary conducting channel, whereas wt CFTR exhibits strong rectification (RIV ≅ 0.4) when it is the primary conducting channel. Conduction through both channels results in an intermediate RIV (≅ 0.6) (6). Holding potential = −40 mV. A and B: each bar, n ≥ 5, *P < 0.05 compared with coexpression with wt CFTR. C: each bar, n = 3, **P < 0.05 compared with myc-SLC26A9 alone.
Fig. 3.
Fig. 3.
SLC26A9 coimmunoprecipitates (co-IPs) immature (band B) CFTR from HEK293 cells coexpressing SLC26A9 with either F508del, wt, or G551D CFTR. A: myc-SLC26A9 also co-IPs mature (band C) CFTR when coexpressed with either wt or G551D CFTR, consistent with an interaction at the plasma membrane. Left panels, IP; right panels, Input. Both the monomeric (~95 kDa) and SDS-resistant dimeric (~190 kDa) forms of myc-SLC26A9 are observed in the input and are immunoprecipitated with myc, as shown. Samples were not treated with forskolin before cell lysis. B: glycosidase assay indicates that myc-SLC26A9 exhibits primarily complex glycosylation when expressed with either wt or F508del CFTR. C = control, P = PNGaseF-treated, and E = EndoH-treated. A 3–8% gradient gel was used for PAGE. C: cell-surface biotinylated myc-SLC26A9 is significantly reduced when coexpressed with F508del CFTR, as compared with wt CFTR, even though total myc-SLC26A9 is not affected. Quantification of biotinylated myc-SLC26A9 includes both monomer and dimer and is normalized by total β-actin; averages from 3 independent experiments, *P < 0.05.
Fig. 4.
Fig. 4.
SLC26A9 coimmunoprecipitates the PDZ-domain proteins CAL and NHERF-1. A: differences in myc-SLC26A9’s ability to co-IP NHERF1 and CAL are dependent on the coexpression of CFTR. Coexpression with wt CFTR increases the co-IP of both PDZ-domain proteins, whereas coexpression with F508del CFTR only affects CAL co-IP. Left panels, IP; right panels, Input. Both monomers and dimers of myc-SLC26A9 are shown. Samples were not treated with forskolin before cell lysis. B: quantification of CAL binding indicates either form of CFTR increases myc-SLC26A9’s ability to co-IP CAL. Normalized by total immunoprecipitated myc-SLC26A9 (monomer plus dimer). Averages from 3 independent experiments, *P < 0.05. C: quantification of NHERF1 binding indicates only coexpression with wt CFTR enhances NHERF1 co-IP. Normalized by total immunoprecipitated myc-SLC26A9 (monomer plus dimer). Averages from 3 independent experiments, *P < 0.05.
Fig. 5.
Fig. 5.
Immunofluorescence demonstrates CFTR-dependent differences in the intracellular localization of SLC26A9. HEK293 cells were cotransfected with myc-SLC26A9 and either wt-GFP-CFTR (A–D) or F508del-GFP-CFTR (E–H). Fixed and permeabilized cells were then immunolabeled with mouse anti-myc (to detect SLC26A9, B and F) and rabbit anti-CAL antibody (C and G). D: the overlay of wt CFTR (A), myc-SLC26A9 (B), and CAL (C) demonstrates colocalization between wt CFTR and myc-SLC26A9 in the plasma membrane region (yellow arrowheads), and in an intracellular compartment that overlaps with CAL (white arrowheads). H: the overlay of F508del CFTR (E), myc-SLC26A9 (F), and CAL (G) demonstrates that myc-SLC26A9 primarily colocalizes with CAL (white arrowheads), and that both F508del CFTR and myc-SLC26A9 are absent at the plasma membrane. Scale bar in D and H, 5 µm. Similar results were observed in 3 biological replicates.
Fig. 6.
Fig. 6.
The increase in CAL co-IP shown in Fig. 4B requires that both SLC26A9 and F508del CFTR have intact PDZ motifs. A: mutation of either myc-SLC26A9’s or F508del CFTR’s PDZ motif at the −2 position threonine abrogates the CFTR-induced increase in CAL co-IP. This mutation does not eliminate the PDZ motif from either protein, although the −2 position Thr is central to PDZ domain binding affinity (17). Left panels, IP; right panels, Input. Both monomers and dimers of myc-SLC26A9 are shown. Samples were not treated with forskolin before cell lysis. B: quantification of CAL binding indicates both proteins require intact PDZ motifs to increase myc-SLC26A9’s ability to co-IP CAL. Normalized by total immunoprecipitated myc-SLC26A9 (monomer plus dimer). Averages from 3 independent experiments, *P < 0.05. C: immature (band B) F508del CFTR co-IPs CAL when expressed alone. Left panels, IP; right panels, Input.
Fig. 7.
Fig. 7.
Mutating the PDZ motif of SLC26A9 restores constitutive current when coexpressed with F508del CFTR. Whole cell patch-clamp measurement of membrane current (Im) observed in HEK293 cells transiently transfected with myc-SLC26A9 ± T789A (PDZ motif mutation) and CFTR. A: constitutive current was measured 1 min after break-in and before any treatments. The indicated myc-SLC26A9 construct was coexpressed with either wt (black bars) or F508del (gray bars) CFTR. B: after measurement of constitutive Im, cells were treated with 10 µM forskolin and the change in Im recorded when the response plateaued (~2 min after forskolin addition). F508del coexpressors did not respond to forskolin (not shown). Holding potential = −40 mV; each bar, n ≥ 5, *P < 0.05 compared with coexpression with wt CFTR. C–D: IF indicates that mutating the PDZ motif of myc-SLC26A9 increases its plasma membrane expression. HEK293cells expressing myc-SLC26A9 ± T789A (PDZ motif mutation) with F508del CFTR were fixed, permeabilized, and labeled with anti-myc antibody to assess distribution of myc-SLC26A9. C: coexpression of myc-SLC26A9 + F508del CFTR. D: coexpression of myc-SLC26A9 T789A + F508del CFTR.
Fig. 8.
Fig. 8.
A model demonstrating potential PDZ domain protein interactions for coexpressed SLC26A9 and CFTR. Left side, F508del CFTR coexpression; right side, wt CFTR coexpression. A: the proteins commence their interaction in the ER, where the CAL dimer binds CFTR and SLC26A9. Misfolded F508del CFTR is removed from the complex and undergoes ERAD, leaving half of the CAL dimer unbound. If SLC26A9 is dimerized at this stage, removal of F508del CFTR may result in a CAL dimer-SLC26A9 dimer complex. A portion of wt CFTR may also be degraded at this stage (see text). Step B: the CAL complex traffics to and through the Golgi complex, either as a CAL-SLC26A9 dimer (F508del CFTR coexpression), or a CAL-SLC26A9-wt CFTR complex. Step C: at the TGN, the CAL-SLC26A9-wt CFTR complex may be disrupted by additional proteins which interact with CFTR, such as TC10 (14) or MAST205 (47). Since an individual SLC26A9 PDZ motif has relatively weak affinity for CAL (Table 1), the complex is released from the TGN and continues trafficking to the recycling endosome and ultimately, plasma membrane. Conversely, the lack of F508del CFTR in the CAL-SLC26A9 dimer complex impedes the recruitment of additional proteins, which, combined with a greater affinity between CAL and SLC26A9 due to avidity, halts the forward trafficking of SLC26A9.

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