Unnatural amino acid (Uaa) technology has broken new ground in the protein research and introduced a plethora of chemical groups that can be incorporated to the existing proteins for new functionality. Here, we incorporated 4,5-dimethoxy-2-nitrobenzyl-Cysteine (Cmn) into the pore region of inwardly rectifying potassium channel Kir2.1 to make a Photo-activatable Inwardly Rectifying Potassium channel, or PIRK. Bulky side chains of Cmn block the channel pore physically, which leaves the channel non-functional. After brief UV irradiation however, 4,5-dimethoxy-2-nitrobenzyl groups are released from Cmn to reactivate the channel. When PIRK was introduced in rat hippocampal primary neurons, the neuronal activity was completely suppressed with a brief UV pulse. We also expressed functional PIRK in the mice embryonic neocortex to present the successful in vivo application of the method. This strategy is directly transferable to other ion channels and receptors, and its application has great potential in unraveling mechanisms of native and diseased brain circuitry. On the other hand, we improved Uaa uptake rate and intracellular concentration in mammalian cells using noncanonical side chains where the Uaa is masked by selected ester groups. This result can boost Uaa incorporation efficiency in mammalian cells, thus reducing the amount of Uaas required

Deciphering neuronal networks governing specific brain functions is a longstanding mission in neuroscience, yet global manipulation of protein functions pharmacologically or genetically lacks sufficient specificity to reveal a neuronal protein's function in a particular neuron or a circuitry. Photostimulation presents a great venue for researchers to control neuronal proteins with high temporal and spatial resolution. Recently, an approach to optically control the function of a neuronal protein directly in neurons has been demonstrated using genetically encoded light-sensitive Unnatural amino acids (Uaas). Here, we describe procedures for genetically incorporating Uaas into target neuronal proteins in neurons in vitro and in embryonic mouse brain. As an example, a photocaged Uaa was incorporated into an inwardly rectifying potassium channel Kir2.1 to render Kir2.1 photo-activatable. This method has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. Uaas with other properties can be similarly incorporated into neuronal proteins in neurons for various applications.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K(+) current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K(+) current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation.