Design of Light-Sensitive NMDARs by Genetically Encoded Photo-Cross-Linkers
Meilin Tian1,2,3,4, Shixin Ye5,6,7,8
1Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai, China.
Abstract:
Genetic code expansion exploiting unnatural amino acids (Uaas) is a powerful technique to create novel protein function in vivo. Here, we provide a protocol for the incorporation of two UV-sensitive crosslinking Uaas into NMDA receptors (NMDARs), a type of glutamate-gated ion channels mediating fast synaptic transmission. Through heterologous expression in Xenopus laevis oocytes, we have identified light-sensitive NMDARs of GluN2B subtype by using the two-electrode voltage electrophysiology measurement in combination with online-UV application. Immunoblotting analysis has been used to confirm inter-subunit crosslinking.
