Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces.
Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.
Ecole Normale Supérieure, Institut de Biologie de l' Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, F-75005 Paris, France.
Shujia
S
Zhu
Morgane
M
Riou
C Andrea
CA
Yao
Stéphanie
S
Carvalho
Pamela C
PC
Rodriguez
Olivier
O
Bensaude
Pierre
P
Paoletti
Shixin
S
Ye
D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006801:Humans; D008027:Light; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D015202:Protein Engineering; D055503:Protein Multimerization / Q000187:drug effects / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D058468:Receptors, Ionotropic Glutamate / Q000737:chemistry / Q000235:genetics*; D014466:Ultraviolet Rays; D014981:Xenopus
D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D021122:Protein Subunits; D058468:Receptors, Ionotropic Glutamate
neurotransmitter receptor; protein structure–function