Improvement of unnatural amino acid technology and its in vivo application via light-activatable potassium channels /

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