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. Thiol-reactive fluorophores are small and bright, but are restricted in terms of the positions on a protein that can be labeled and present significant issues with background incorporation. Genetically encoded fluorescent protein tags are specific to a protein of interest, but are very large and usually only used to label the free N- and C-termini of proteins. L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP) is a fluorescent amino acid that can be specifically incorporated into virtually any site on a protein of interest using amber stop-codon suppression. Due to its environmental sensitivity and potential as a donor in fluorescence resonance energy transfer experiments, it has been adopted by numerous investigators to study voltage, ligand, and temperature-dependent activation of a host of ion channels. Simultaneous measurements of ionic currents and ANAP fluorescence yield exceptional mechanistic insights into channel function. In this chapter, I will summarize the current literature regarding ANAP and ion channels and discuss the practical aspects of using ANAP, including potential pitfalls and confounds.