Monomeric CFTR in Plasma Membranes in Live Cells Revealed by Single Molecule Fluorescence Imaging

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel. There is indirect and conflicting evidence about whether CFTR exists in cell membranes as monomers, dimers, or higher order oligomers. We measured fluorescence intensities and photobleaching dynamics of distinct fluorescent spots in cells expressing functional CFTR-green fluorescent protein (GFP) chimeras. Intensity analysis of GFP-labeled CFTR in live cells showed singlecomponent distributions with mean intensity equal to that of purified monomeric GFP, indicating monomeric CFTR in cell membranes. Fluorescent spots showed single-step photobleaching, independently verifying that CFTR is monomeric. Results did not depend on whether GFP was added to the CFTR N terminus or fourth extracellular loop or on whether CFTR chloride conductance was stimulated by cAMP agonists. Control measurements with a CFTR chimera containing two GFPs showed two-step photobleaching and a single-component intensity distribution with mean intensity twice that of monomeric GFP. These results provide direct evidence for monomeric CFTR in live cells.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is a member of the ATP-binding cassette protein family that forms cAMP-regulated chloride channels (1). CFTR is expressed in epithelial cells in the airways, pancreas, intestine, and other tissues (2). Loss-of-function mutations in CFTR cause the hereditary lethal disease cystic fibrosis, in which chronic lung infection produces morbidity and mortality (1, 2). Excessive CFTR activity in the intestine in response to bacterial enterotoxins produces secretory diarrheas (3, 4). There is considerable interest in CFTR structure and assembly in cell membranes as CFTR is an important drug target for therapy of cystic fibrosis, secretory diarrheas, and polycystic kidney disease (3, 5-7).The assembly state of CFTR has been controversial, with indirect evidence reported for CFTR monomers, dimers, and mixed monomers/dimers. Patch clamp analysis of constructs containing linked wild-type (WT) CFTRs or WT and mutant CFTRs suggested that two CFTR polypeptides form a single chloride conductance pathway (8). Conflicting data from reconstituted membranes containing WT and mutant CFTRs did not reveal intermediary conductance states, consistent with independently functioning CFTR monomers (9). Electron crystallography has indicated that CFTR is a monomer with two conformations, likely the open and closed channel states (10). These data are in accord with high resolution crystal structures of bacterial ATP-binding cassettetype transporters showing unit cells containing two transmembrane-nucleotide binding domains (11). Biochemical approaches including velocity-gradient centrifugation, coimmunoprecipitation, gel filtration, and cross-linking have generated conflicting data suggesting monomeric CFTR (9, 12), dimeric CFTR (13), and mixed monomeric/dimeric CFTR (14, 15). Data supporting dimeric CFTR have also come from patch clamp of CFTR in the presence of...

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ABCA1 dimer–monomer interconversion during HDL generation revealed by single-molecule imaging

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Aquaporin-4 (AQP4) Associations and Array Dynamics Probed by Photobleaching and Single-molecule Analysis of Green Fluorescent Protein-AQP4 Chimeras

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“…Images were processed, and backgroundsubtracted area-integrated intensities were analyzed as described previously (21). Time-lapse images of AQP4 OAPs in live cells were acquired at three frames/min for up to 3 h, with the excitation laser shuttered between image acquisitions.…”