A Sensitive Tool to Measure CFTR Channel Activity Katalin Goda*, G abor Szal oki  Key terms CFTR; yellow fluorescent protein; flow cytometry; chloride channel activity THE cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7) is a member of the human ABC protein fam- ily. The characteristic feature of ABC proteins is the similar architecture of the ATP binding sites and the common mecha- nism of ATP binding and hydrolysis. In contrast to most of the ABC proteins, CFTR is not an active transporter, rather it is a phosphorylation and ATP hydrolysis gated chloride ion channel and channel regulator (1,2). Mutations in both copies of the CFTR gene may lead to a heritable genetic disease, i.e. cystic fibrosis (CF) with variable severity depending on the site of the mutation. Certain mutations alter biogenesis or cel- lular processing of the CFTR protein, while others affect its channel activity. Chemical chaperons, also called correctors (e.g., VX-809) can partially rescue the misprocessing, most likely by improving the folding of the protein at the endoplas- mic reticulum (2). Activators of CFTR may function through elevating cytosolic cAMP (promoting CFTR phosphoryla- tion), inhibiting phosphatase activity (thus blocking CFTR de- phosphorylation), and/or interacting directly with the channel protein (1). Since CFTR is expressed in the epithelial cells of several organs, CF is a multi-organ disease that affects the sinopulmonary and male urogenital systems, alters pancreatic and biliary secretion, therefore the current average lifespan of CF patients is approximately 40 years of age (1,2). In most cases, the primary cause of early onset of death is the progres- sion of lung disease (2,3). The mechanisms by which CFTR mutations cause lung disease in CF patients are not fully understood. It may include altered ion and water transport across the airway epithelium and aberrant inflammatory and immune responses to pathogens within the airways (3,4). Several techniques have been developed to study CFTR function or to search for CFTR activators or modulators including halide sensitive fluorescent dyes, electrophysiologi- cal approaches such as patch clamp, short-circuit measure- ments in Ussing chambers and influx or efflux measurements applying radioactive ions. In addition to the above techniques a halide sensitive mutant form of yellow fluorescent protein (YFP) has been also utilized to probe CFTR function meas- uring the iodide-mediated quenching rate of YFP, since the open state of CFTR is also permeable to iodide ions (5). YFP fluorescence intensity is both dependent on the intracellular iodide concentration and YFP expression level. Therefore, flu- orescence intensities measured in the presence of iodide should be normalized to YFP fluorescence intensities meas- ured in the absence of iodide to decrease experimental vari- ability related to the differences in sensor expression level among individual cells. However, normalization to the un-quenched YFP fluores- cence intensity is problematic especially in flow cytometric assays. The co-expression of a halide insensitive fluorescent protein can overcome the above problem as was demonstrated by Vijftigschild et al. published in the current issue of Cytome- try Part A (page 576). When the two fluorescent proteins were expressed as a fusion protein formation of aggregates was observed. To avoid this problem the two proteins were con- nected by an auto-cleavable polypeptide chain (6) resulting in physically not connected proteins. Theoretically, the expres- sion level of the two proteins may change upon time due to Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary Received 28 January 2013; Revision Received 20 February 2013; Accepted 5 March 2013 Grant sponsor: OTKA; Grant number: PD75994. Szodoray Fellowship. *Correspondence to: Katalin Goda, Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary. E-mail: goda@med.unideb.hu Published online 4 April 2013 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/cyto.a.22290 © 2013 International Society for Advancement of Cytometry COMMENTARY Cytometry Part A • 83A: 528529, 2013