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 photo-reactive 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 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 GluN2B, than GluN2A, NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype-specificity through the genetic code expansion.
Inhibition sculpts neural activity through various cell types and circuits, but, unlike excitation, it is not self-propagating and must be locally recruited with a temporal delay. Here the authors show a fast, feedforward inhibitory mechanism that bypasses synaptic delay through ephaptic coupling of an interneuron to the axon initial segment of a projection cell.
Le magazine La Recherche, créé il y a plus de 40 ans, s'efforce de rendre accessible au plus grand nombre les travaux de recherches publiés à travers la planète. S'adressant à un lectorat d'experts passionnés autant que d'amateurs éclairés, La Recherche reste ouvert à toutes les sciences et à toutes les technologies dans leurs dimensions de découverte et d'approfondissement des savoirs nouveaux.
C'est pourquoi le magazine a créé il y a neuf ans le Prix La Recherche, remis chaque année afin de suivre au plus près les avancées de la science.
Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons ; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution.