The synthesis of fluorescent unnatural amino-acid Anap was optimized and the Anap was incorporated into four sites in an acid-pocket or a transmembrane region of human acid-sensing ion channel-1a (hASIC1a). Combinational Anap fluorescence spectra and patch-clamp electrophysiology data illustrated site-specific conformational responses upon toxin mambalgin-1 binding. This combinational approach can be used to analyse conformational properties of many different eukaryotic proteins in their functional states, in a site-specific manner in live mammalian cells.