Abstract
We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.
Highlights
Several important classes of protein are directly regulated by ligand binding
These range from soluble enzymes to membrane-embedded proteins including receptor tyrosine kinases, G protein-coupled receptors (GPCRs), and ion channels
We have developed a Förster resonance energy transfer (FRET)-based assay for the measurement of adenine nucleotide binding to membrane proteins in real time
Summary
Förster resonance energy transfer (FRET) is a method that detects the proximity between two fluorescently tagged molecules[3 ]. We measure nucleotide binding as FRET between ANAP-labeled protein and fluorescent, trinitrophenyl (TNP) nucleotide derivatives (Figure 1A). Nucleotide binding to the intracellular side of ANAP-labeled KATP channels is measured in cells that have been unroofed by sonication leaving adherent fragments of plasma membrane on a glass cover slip10 , 11 , 13 , 14. Nucleotide binding to ANAP-labeled KATP channels is measured in a membrane patch under voltage clamp, allowing for the simultaneous measurement of ionic currents and fluorescence. By combining these two experimental approaches, changes in binding can be directly correlated with changes in channel function[11 ].
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