• 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 family. The characteristic feature of ABC proteins is the similar architecture of the ATP binding sites and the common mechanism 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 cellular 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 endoplasmic reticulum (2). Activators of CFTR may function through elevating cytosolic cAMP (promoting CFTR phosphorylation), inhibiting phosphatase activity (thus blocking CFTR dephosphorylation), 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 progression 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, electrophysiological approaches such as patch clamp, short-circuit measurements 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 measuring 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, fluorescence intensities measured in the presence of iodide should be normalized to YFP fluorescence intensities measured in the absence of iodide to decrease experimental variability related to the differences in sensor expression level among individual cells.

However, normalization to the un-quenched YFP fluorescence 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 Cytometry 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 connected by an auto-cleavable polypeptide chain (6) resulting in physically not connected proteins. Theoretically, the expression level of the two proteins may change upon time due to different turnover rates; however, significant deviations are unlikely during the short time course of the measurements. In their experiments, the authors used Venus, the brighter version of YFP, and found that the Venus/mKate fluorescent protein pair suits best for flow cytometric measurements, due to the slower quenching rate of Venus upon addition of iodide. Normalization to the fluorescence intensity of the iodide insensitive protein (mKate) corrects for the differences in sensor expression level during the experiment and thereby reduces experimental variability and improves accuracy. The normalized quenching rates of individual cells (monitored by microscopy) or cell populations (measured by flow cytometry) were independent of sensor expression levels further validating the reliability of their method.

Previously, the ratiometric sensor Clomeleon applying fluorescence resonance energy transfer (FRET) had been used to measure intracellular chloride concentrations (7). Clomeleon is a fusion protein consisting of CFP and YFP, joined by a flexible 24-amino acid linker. The CFP/YFP pair is one the most popular fluorescent protein pair for FRET analysis (see, e.g. Refs.8, 9), owing to the facts that both the excitation spectra and emission spectra of the two fluorophores are sufficiently separated promoting their separate excitation and detection. Unlike other FRET-based indicator proteins (e.g., the Ca2+-sensitive sensors Cameleon and Pericam (10)) Clomeleon does not require a conformational change between the units of the FRET pair, as quenching of YFP by Cl is sufficient to induce a change in the YFP/CFP emission ratio. Thus, exciting the CFP donor causes the YFP acceptor to be excited and to emit yellow fluorescence. Binding Cl to YFP quenches the yellow fluorophore (11) decreasing the degree of FRET. Thus, measuring the emission ratio of YFP relative to that of CFP (while exciting CFP) provides an absolute measure of the intracellular Cl concentration (12). The disadvantage of Clomeleon is that it cannot be used in most flow cytometers, because of the lack of appropriate excitation wavelength for CFP. In contrast to Clomeleon the spectral characteristics of the Venus/mKate pair are favorable for flow cytometry suggesting a preferential use of this pair in future applications (Vijftigschild et al., page 576).

Genetically encoded fluorescent optical probes have become powerful tools for visualization of ions and ion channel activities in live cells by fluorescence microscopy, fluorescence plate reader, or flow cytometry. Appropriate normalization for alterations in sensor expression using ratiometric sensors like the Venus/mKate pair in the case of chloride flux measurements can make this technique applicable on flow cytometers.

Recent data indicate that CFTR deficiency of the immune cells may also contribute to the increased inflammatory responses occurring in CF (3, 4). The ratiometric assay can be a valuable tool to measure CFTR function in case of non-adherent cells, such as immune cells, contributing to the detailed understanding of this life-threatening disease. In addition, this ratiometric method could be applied in drug screening assays searching for more powerful CFTR corrector or activator compounds with therapeutic benefit.

Literature Cited

  1. Top of page
  2. Literature Cited