CFTR potentiators partially restore channel function to A561E‐CFTR, a cystic fibrosis mutant with a similar mechanism of dysfunction as F508del‐CFTR

Abstract

Background and Purpose Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel causes the genetic disease cystic fibrosis (CF). Towards the development of transformational drug therapies for CF, we investigated the channel function and action of CFTR potentiators on A561E, a CF mutation found frequently in Portugal. Like the most common CF mutation F508del, A561E causes a temperature‐sensitive folding defect that prevents CFTR delivery to the cell membrane and is associated with severe disease. Experimental Approach Using baby hamster kidney cells expressing recombinant CFTR, we investigated CFTR expression by cell surface biotinylation, and function and pharmacology with the iodide efflux and patch‐clamp techniques. Key Results Low temperature incubation delivered a small proportion of A561E‐CFTR protein to the cell surface. Like F508del‐CFTR, low temperature‐rescued A561E‐CFTR exhibited a severe gating defect characterized by brief channel openings separated by prolonged channel closures. A561E‐CFTR also exhibited thermoinstability, losing function more quickly than F508del‐CFTR in cell‐free membrane patches and intact cells. Using the iodide efflux assay, CFTR potentiators, including genistein and the clinically approved small‐molecule ivacaftor, partially restored function to A561E‐CFTR. Interestingly, ivacaftor restored wild‐type levels of channel activity (as measured by open probability) to single A561E‐ and F508del‐CFTR Cl− channels. However, it accentuated the thermoinstability of both mutants in cell‐free membrane patches. Conclusions and Implications Like F508del‐CFTR, A561E‐CFTR perturbs protein processing, thermostability and channel gating. CFTR potentiators partially restore channel function to low temperature‐rescued A561E‐CFTR. Transformational drug therapy for A561E‐CFTR is likely to require CFTR correctors, CFTR potentiators and special attention to thermostability.

Partial rescue of F508del‐cystic fibrosis transmembrane conductance regulator channel gating with modest improvement of protein processing, but not stability, by a dual‐acting small molecule

“…To explore the specificity of CFFT‐004, we tested its effects on A561E, another class II‐III‐VI CF mutation (Veit et al, ) found in NBD1 with defects closely comparable to those of F508del‐CFTR (Mendes et al, ; Wang et al, ). Figures and demonstrate that when BHK cells expressing either F508del‐ or A561E‐CFTR were grown at 37°C, no mature protein was produced and no channel activity was observed.…”

“…In (A) and (B), some data were originally published in Wang et al . () (A, n = 4; B, n = 5); other data are newly acquired. Other details as in Figure .…”

“…With the Ussing chamber and patch‐clamp techniques, we analysed the effects of correction and potentiation with CFFT‐004 on the plasma membrane stability and channel gating defects of F508del‐CFTR. To probe the mechanism of action of CFFT‐004, we used the F508del‐CFTR revertant mutations G550E and R1070W (deCarvalho et al, ; Thibodeau et al, ), and to explore specificity, we studied A561E, another class II‐III‐VI CF mutation (Veit et al, ) with defects closely comparable to those of F508del‐CFTR (Mendes et al, ; Wang et al, ). Our results demonstrate that CFFT‐004 has differential effects on F508del‐CFTR, but fails to rescue A561E‐CFTR.…”

Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function

“…Figure 4C shows the mean of current densities at 0 mV during stimulation of F508del-CFTR by Fsk + Gst at 22°C ( n = 6) or 35°C ( n = 5). According to the literature (Wang et al, 2011, 2014; Liu et al, 2012) and contrary to the CFTR response obtained at 22°C, only a small transient current is recorded at physiological temperature. These results show that whole-cell CFTR currents can be studied at 37°C using APC, allowing drug effects to be studied under physiological conditions.…”

A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis‐causing mutation

“…1 and 3). As described in the Materials and Methods, we made prolonged, uninterrupted recordings at room temperature, at which ∆F508‐CFTR channel activity is stable ( e.g ., 44), to accurately determine channel number and acquire sufficient transitions to quantify channel gating. The single‐channel current amplitude ( i ) of these 4 deletion mutants is similar to that of WT CFTR (Fig.…”