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The effect of adenosine (ADO) on fluorescence resonance energy transfer (FRET) between CFTR and the A2B receptor (A2B‐R)
Author(s) -
Watson Michael J,
Rollins Brett,
Tarran Robert
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a424-d
Subject(s) - förster resonance energy transfer , chemistry , transfection , biophysics , extracellular , receptor , fluorescence , microbiology and biotechnology , biochemistry , biology , physics , quantum mechanics , gene
The A2B‐R has previously been shown to activate CFTR in airway epithelia leading us to infer that ADO is an important regulator of airway surface liquid (ASL) volume. However, ASL [ADO], as measured by lavage is ~100 nM, a concentration too low to activate A2B‐R which typically have an EC 50 of ~2 μM. Accurate measurements of ASL [ADO] are difficult due to the low volumes of ASL present on airway surfaces and we may be underestimating [ADO] in the microenvironment at the cell surface. Thus, we have designed a fluorescent resonance energy transfer (FRET) pair of cfp‐CFTR (labeled at the n‐terminus) and yfp‐A2B‐R (labeled at the c‐terminus) that is sensitive to the extracellular ADO concentration to measure cell surface ADO. Both of the constructs were shown to be functional using iodide efflux and ADO stimulated cAMP accumulation respectively. We evaluated the ADO sensitivity of this FRET pair transfected into BHK‐21 cells. ADO increased FRET in a dose‐dependent manner with an EC 50 of 1.5μM (n=6). An A2B‐R construct where yfp was attached to the n‐terminus was also tested. The n‐terminus of the A2B‐R is extracellular and in this case no FRET was detected. Thus we predict that this FRET pair will be a useful tool in measuring ASL [ADO]. We are currently focusing our efforts on placing these constructs in a retroviral expression system in order to express them in well‐differentiated airway epithelia. Supported by NHLBI RO1 HL074158 The effect of adenosine (ADO) on fluorescence resonance energy transfer (FRET) between CFTR and the A2B receptor (A2B‐R)