ANAP: A versatile, fluorescent probe of ion channel gating and regulation.

Fluorescence spectroscopy and microscopy are non-destructive methods that provide real-time measurements of ion channel structural dynamics. As such, they constitute a direct path linking the high-resolution structural models from X-ray crystallography and cryo-electron microscopy with the high-resolution functional data from ionic current measurements. The utility of fluorescence as a reporter of channel structure is limited by the palette of available fluorophores.

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time.

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.

Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry.

Pancreatic ATP-sensitive K channels (K) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.

Activation mechanism of ATP-sensitive K channels explored with real-time nucleotide binding.

The response of ATP-sensitive K channels (K) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of K in channel gating. Binding to NBS2 was Mg-independent, but Mg was required to trigger a conformational change in SUR1.