Abstract
The most common defect in cystic fibrosis, deletion of phenylalanine from position 508 of the cystic fibrosis transmembrane conductance regulator (Delta F508 CFTR), decreases the trafficking of this protein to the cell surface membrane. Previous studies have shown that low temperature and high concentrations of glycerol or trimethylamine N-oxide can partially counteract the processing defect of Delta F508 CFTR. The present study investigates whether physiologically relevant concentrations of organic solutes, accumulated by cotransporter proteins, can rescue the misprocessing of Delta F508 CFTR. Myoinositol alone or myoinositol, betaine, and taurine given sequentially increased the processing of core-glycosylated, endoplasmic reticulum-arrested Delta F508 CFTR into the fully glycosylated form of CFTR in IB3 cells or NIH 3T3 cells stably expressing Delta F508 CFTR. Pulse-chase experiments using transiently transfected COS7 cells demonstrated that organic solutes also increased the processing of the core-glycosylated form of green fluorescent protein-Delta F508 CFTR. Moreover, the prolonged half-life of the complex-glycosylated form of GFP-Delta F508 CFTR suggests that this treatment stabilized the mature form of the protein. In vitro studies of purified NBD1 stability and aggregation showed that myoinositol stabilized both the Delta F508 and wild type CFTR and inhibited Delta F508 misfolding. Most significantly, treatment of CF bronchial airway cells with these transportable organic solutes restores cAMP-stimulated single channel activity of both CFTR and outwardly rectifying chloride channel in the cell surface membrane and also restores a forskolin-stimulated macroscopic 36Cl- efflux. We conclude that organic solutes can repair CFTR functions by enhancing the processing of Delta F508 CFTR to the plasma membrane by stabilizing the complex-glycosylated form of Delta F508 CFTR.
Highlights
The most common mutation leading to cystic fibrosis (CF),1 a deletion of phenylalanine at position 508 in the cystic fibrosis
These results demonstrate that treatment with organic solutes can rescue the processing defect of ⌬F508 CFTR in the CF bronchial airway IB3 cell line as assessed by the glycosylation state
The studies presented here demonstrate that myoinositol alone, or myoinositol given sequentially with taurine and betaine, can increase the processing of the core-glycosylated, endoplasmic reticulum-arrested ⌬F508 CFTR into the fully glycosylated mature form of CFTR, in both IB3 cells, a CF airway cell line, NIH 3T3 fibroblasts stably transfected with ⌬F508 CFTR, and COS7 cells transiently transfected with GFPtagged ⌬F508 CFTR
Summary
The most common mutation leading to cystic fibrosis (CF), a deletion of phenylalanine at position 508 in the cystic fibrosis. The major renal organic solutes such as 300 mM sorbitol, myoinositol, and taurine recently have been shown to increase processing in renal medulla cells transfected with ⌬F508 CFTR [15]. Together, these observations suggest the potential utility of treating airways with organic solutes or “chemical chaperones” for CF therapy. Myoinositol Facilitates ⌬F508 CFTR Activity data suggesting that organic solutes can be used in much lower concentrations than previously suspected, by utilizing the activity of endogenous organic solute cotransporters that actively accumulate these solutes These accumulated solutes correct the chloride channel function of CFTR and promote
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