gamma-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core

Background: The AMPA-type ionotropic glutamate receptor mediates fast excitatory neurotransmission in the brain. A variety of auxiliary subunits regulate its gating properties, assembly, and trafficking, but it is unknown if the binding of these auxiliary subunits to the receptor core is dynamically regulated. Here we investigate the interplay of the two auxiliary subunits gamma-2 and GSG1L when binding to the AMPA receptor composed of four GluA1 subunits. Methods: We use a three- color single-molecule imaging approach in living cells, which allows the direct observation of the receptors and both auxiliary subunits. Colocalization of different colors can be interpreted as interaction of the respective receptor subunits. Results: Depending on the relative expression levels of gamma-2 and GSG1L, the occupancy of binding sites shifts from one auxiliary subunit to the other, supporting the idea that they compete for binding to the receptor. Based on a model where each of the four binding sites at the receptor core can be either occupied by.-2 or GSG1L, our experiments yield apparent dissociation constants for gamma-2 and GSG1L in the range of 2.0-2.5/mu m2. Conclusions: The result that both binding affinities are in the same range is a prerequisite for dynamic changes of receptor composition under native conditions. Keywords: Receptor assembly, Subunit stoichiometry, AMPA receptor regulatory subunits, Single-molecule imaging

Automated summary

Using single-molecule imaging, this study investigates the binding of auxiliary subunits gamma-2 and GSG1L to the AMPA receptor. The results show that both subunits compete for binding sites on the receptor and have similar binding affinities, suggesting dynamic changes in receptor composition under native conditions.