A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator

Abstract

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (DF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct DF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC DF508 CFTR biogenesis and function. C4 stabilized I507-ATT DF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC DF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC DF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC DF508 CFTR. Our results suggest that binding of C4 and VX-809 to DF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.

Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis

“…Single cell clones of human embryonic kidney 293 (HEK-293) cells stably expressing WT CFTR, I507-ATT or I507-ATC ΔF508 CFTR were developed and maintained as described [ 22 – 24 ].…”

“…CFTR stability was determined following cycloheximide block (0.2 mg/ml) using following formula: half-life equals elapsed time multiplied by logarithm of two, divided by logarithm of ratio of beginning amount and ending amount (t 1/2 = (elapsed time * log2) / log (beginning amount/ending amount)), as described [ 23 , 24 ]. Cells were pretreated with CFTR correctors/potentiators for 16 hours and the compounds were present during the chase period.…”

“…We have demonstrated that the I507-ATC/ATT synonymous codon change contributes to the misfolding [ 22 ] and functional defects of ΔF508 CFTR [ 23 ]. In a follow up study, we determined that while VX-809 corrected both variants, the beneficial effects of the bisaminomethylbithiazole compound Corr-4a were specific to the native, I507-ATT ΔF508 [ 24 ]. We have also demonstrated that Corr-4a+VX809 combination enhanced rescue efficiency [ 24 ].…”

“…In a follow up study, we determined that while VX-809 corrected both variants, the beneficial effects of the bisaminomethylbithiazole compound Corr-4a were specific to the native, I507-ATT ΔF508 [ 24 ]. We have also demonstrated that Corr-4a+VX809 combination enhanced rescue efficiency [ 24 ]. Considering these results, we hypothesized that stabilization of ΔF508 CFTR band B with Corr-4a counteracts the negative effects of VX-770 on VX-809-rescued ΔF508 CFTR.…”

Why should mitochondria define species?

“…Synonymous codons may also modulate protein folding or membrane insertion concomitant with translation [51], as suggested for synonymous codons that modulate phenotypes of the cystic fibrosis transmembrane conductance regulator [52] or the pglycoprotein (multidrug resistance protein) [53]. The cited cases involve cytoplasmic translation of bacteria, bacteriophage, and drosophila nuclear genes.…”