Tue, 15/07/2014 - 14:15
Campus SB Geb. E2 6 Raum E.04

Dr. Shixin Ye-Lehmann
Host: Albrecht Ott
Dept de Biologie, Ecole Normale Supérieure, Paris

Genetically encoding light sensitive cross-linkers in glutamate receptors to probe subunit interface

Site-directed mutagenesis with unnatural amino acid (UAA) has been an important methodological advance in molecular biology. In particular, incorporating photoactive UAAs in ligand-gated ion channels provides a powerful way to design light-controlled receptors. Here we demonstrate the feasibility of genetically encoding UAAs in N-methyl-D-aspartate receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in brain function. Using p-Azido-Phenylalanine (AzF) and p-Benzoyl-Phenylalanine (Bpa) as photocrosslinkers, we successfully engineered light-sensitive NMDARs. We show that the incorporation of photo-cross-linkers at the GluN1/GluN2B N-terminal domain (NTD) dimer interface, a region distal to the transmembrane gating machinery, leads to a specific and irreversible inhibition of receptor activity upon UV illumination. Surprisingly, light-dependent inactivation is not observed in GluN1/GluN2A receptors incorporating the photocrosslinker at a homologous position. Our results identify the GluN1/GluN2 NTD dimer interface as a critical structural determinant that dictates UV-controlled inactivation in a subunit-specific manner. We suggest that the NTD dimer association in GluN1/GluN2A receptors has fewer structural constraints (i.e. forming a looser complex) than in GluN1/GluN2B receptors. Our work provides novel information on the importance of subunit interfaces in the subunit-specific regulation of receptor activity. It also paves the way for the design of light-controlled ligand-gated ion channels with subtype-specificity using the technical simplicity afforded by the genetic code expansion.

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