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.

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.

ABSTRACTmembrane sculpts the membrane into a vesicle for cargo transport from the ER to the cis Golgi.