Eric Hosy, Daniel Choquet et coll. inNeuron

Glutamate-Induced AMPA Receptor Desensitization Increases Their Mobility and Modulates Short-Term Plasticity through Unbinding from Stargazin
Audrey Constals, Andrew C. Penn, Benjamin Compans, Estelle Toulme,  Amandine Phillipat,
Sebastien Marais, Natacha Retailleau, Anne-Sophie Hafner, Francoise Coussen, Eric Hosy, and Daniel Choquet,
University of Bordeaux, Interdisciplinary Institute for Neuroscience, France , UMR 5297, Bordeaux Imaging Center, INSERM, 
Neuron 85, 1–17, February 18, 2015 a2015 Elsevier Inc.

Glutamate receptor conformation regulates their mobility to fine tune synaptic transmission

AMPA receptors are tetrameric ion channels gated by glutamate that mediate the majority of excitatory neurotransmission. Their mobility was discovered in Bordeaux more than a decade ago and postulated to tune frequency dependent synaptic transmission. However, how could AMPA receptors exchange within tens of milliseconds in front of glutamate release sites has remained mysterious and intrigued physiologists in the last years. This study now provides a molecular mechanism to explain the interplay between AMPA receptor dynamic organization, activation and synaptic transmission. In a multidisciplinary approach associating many of the members of the team “Dynamic Organization of Synapses” at the Interdisciplinary Institute for Neuroscience, Daniel Choquet and Eric Hosy have co-supervised a study combining superresolution imaging, cell biology, biochemistry and electrophysiology to unravel the impact of AMPA receptor conformation on their surface trafficking

What is the background of this study?
AMPA subtype of glutamate receptors (AMPAR) are concentrated in the post-synapse, in front of glutamate release sites in small domains called nanodomains that we characterized two years ago using superresolution imaging. AMPAR do not come alone, but are associated with a variety of auxiliary proteins such as Stargazin that allows their stabilization in the post-synaptic density by interaction with scaffold proteins such as PSD95.

 What are the properties of basal synaptic transmission?   Upon electric stimulation, glutamate is released in the synaptic cleft and activates AMPA receptors that become open for a few milliseconds, allowing ion flow. Rapidly, activated receptors become desensitized and this leads to a decrease in post-synaptic current. This desensitized state is stable for tens of milliseconds. This creates a refractory period during which receptors cannot be re-activated, thus shaping the frequency dependent response of synapses in a process called short term plasticity.  When a second stimulus occurs within tens of milliseconds of the first one, only a small number of receptors are available for activation, hence the second response is much decreased in comparison to the first one.

The role of AMPAR mobility in tuning short term plasticity
The high proportion of mobile AMPAR in the synapse has a strong impact on short term plasticity as it accelerates recovery from short term depression. In a synapse with mobile receptors, activation and receptor desensitization proceeds similarly, except that desensitized receptors can be exchanged by naïve ones so that when a second stimulus comes in, there are more receptors available for activation. Thus the second response is larger when AMPAR are mobile.

An important question in the field
AMPAR are quite stable in nanodomains while they are mobile in-between them. As most likely post-synaptic currents are largely mediated by clustered receptors, how to reconcile the relative stability of receptors inside clusters with their fast turnover necessary to impact short plasticity?

Desensitized AMPAR become mobile through unbinding from Stargazin.
Our working hypothesis has been that glutamate increases transiently AMPAR mobility. To investigate this question, we first measured the variation of AMPAR mobility induced by glutamate with super-resolution imaging techniques. Individual AMPAR visited a larger area in the presence of glutamate, suggesting that glutamate binding to AMPAR leads to their increased mobility. We thus investigated which AMPAR conformational state might display an increased mobility.

We used pharmacology and expressed various AMPAR mutants locked in specific conformations. We used various point mutations that lock receptors in the closed, open or desensitized state. The quantification clearly demonstrates that desensitized receptors present a higher mobility than closed or opened one. Which mechanisms could explain such increases of the mobility? Changes in mobility imply unbinding from a molecular trap. This molecular unbinding can happen either between the cytoskeleton and scaffolding proteins, or between scaffolding protein and auxiliary protein or between auxiliary proteins and AMPAR.

To answer that question, we performed biochemical experiments, and we showed that stargazin, one of the main auxiliary protein, binds less to desensitized receptor than to closed ones. Finally we estimated the effect of this glutamate induced mobility of desensitized receptors on synaptic transmission. To this aim, we expressed either wild type AMPA receptor and stargazin or a genetic fusion of these two proteins. The prevention of the capacity of AMPA receptor-stargazin to unbind leads to an important synaptic depression.

What could be the molecular mechanism of this dissociation between Stargazin and AMPA receptors?
Recent work from several labs has shown that AMPA receptor desensitization is associated with major conformational changes up to a complete separation of the amino terminal extracellular domains. Our model is thus that this large conformational changes upon desensitization induce a weakening of the AMPAR-Stargazin interaction that allows AMPAR to diffuse out of the stargazin-PSD anchoring sites.

In conclusion, we have shown that

• Glutamate increases AMPAR diffusion in a conformation-dependent manner
• Desensitized AMPAR diffuse faster than closed/resting or open ones
• Desensitized AMPAR dissociate from stargazin
• Glutamate induced AMPAR mobility speeds recovery from synaptic short-term depression