Design of Light-Sensitive NMDARs by Genetically Encoded Photo-Cross-Linkers

Methods Mol Biol. 2017:1677:185-197. doi: 10.1007/978-1-4939-7321-7_10.

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.

Keywords: Genetic code expansion; Photo-cross-linkers; Xenopus laevis oocytes; p-Azido-L-phenylalanine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / chemistry
  • Animals
  • Electrophysiology
  • Oocytes / metabolism
  • Phenylalanine / chemistry
  • Receptors, N-Methyl-D-Aspartate / chemistry*
  • Receptors, N-Methyl-D-Aspartate / metabolism*
  • Xenopus laevis

Substances

  • Amino Acids
  • Receptors, N-Methyl-D-Aspartate
  • Phenylalanine