Site-specific incorporation of unnatural amino acids has wide-ranging applications in the structure-function studies of proteins, offering high specificity, arbitrary environmental sensitivity of incorporation and a nearly unlimited variety of unnatural side chains. We have used photo-activatable unnatural amino acids to probe the activation and desensitization of glutamate receptors (GluRs), ligand-gated ion channels that mediate fast excitatory synaptic transmission in the brain. We introduced the unnatural amino acid cross-linker p-Benzoyl-Phenylalanine (Bpa) to the AMPA receptor GluA2 expressed in mammalian cell lines. Sites of interest were replaced by an amber stop codon (TAG). The stop codon was suppressed using a genetically-encoded exogenous tRNA and tRNA-synthetase pair that incorporate Bpa (Ye et al, 2008 J Biol Chem). We initially chose sites in the lower (D2) lobes of the LBD (S729, G725), which have been previously shown to form intermolecular disulfide cross-links that inhibit the receptor (Armstrong et al, 2006 Cell; Plested and Mayer, 2009 J. Neurosci). Patch clamp electrophysiology coupled to rapid perfusion demonstrated that Bpa-rescued GluRs had similar properties to wild-type GluRs. Receptors could be trapped in inactive conformational states upon UV irradiation and subsequent photo-cross-linking, consistent with previous work. Irreversible trapping was characterized by near-complete peak current reduction (95 %) in less than five seconds cumulative exposure to UV at 365 nm from a mercury lamp. The cross-linking rate depended on UV intensity and exposure time. Wild-type channels were unaffected by similar UV exposures. Our results demonstrate that genetically-encoded unnatural amino acids such as Bpa provide an convenient strategy to control structural rearrangements of ligand-gated ion channels in mammalian cells.