Abstract
The subunit GluR2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subfamily of ionotropic glutamate receptors (GluRs) features a single amino acid at the narrow constriction of the pore loop that is altered from glutamine to arginine by RNA editing. This so-called Q/R site has been shown to play an important role in the determination of the electrophysiological properties of AMPA receptor complexes as well as of trafficking to the plasma membrane. The protein stargazin has also been shown to modulate electrophysiological properties and trafficking to the plasma membrane of AMPA receptors. In this study we examined via a series of mutants of the Q/R site of the AMPA receptor GluR1 whether the amino acid at this position has any influence on the modulatory effects mediated by stargazin. To this end, we analyzed current responses of Q/R site mutants upon application of glutamate and kainate and determined the amount of mutant receptor protein in the plasma membrane in Xenopus oocytes. Desensitization kinetics of several mutants were analyzed in HEK293 cells. We found that the stargazin-mediated decrease in receptor desensitization, the slowing of desensitization kinetics, and the kainate efficacy were all dependent on the amino acid at the Q/R site, whereas the stargazin-mediated increase in trafficking toward the plasma membrane remained independent of this amino acid. We propose that the Q/R site modulates the interaction of stargazin with the transmembrane domains of AMPA receptors via an allosteric mechanism and that this modulation leads to the observed differences in the electrophysiological properties of the receptor.
Highlights
The majority of the excitatory synaptic transmission in the mammalian central nervous system is mediated by AMPA4 receptors (AMPARs)
As it is known that Q/R editing site of AMPA receptors is an important determinant of receptor complex assembly, trafficking, [7] and several electrophysiological properties [2,3,4,5], we examined whether and how different amino acids at the Q/R site influence the modulatory effects of stargazin on the AMPA receptor complex
It has already been shown that both stargazin and the amino acid at the Q/R site of AMPARs, independently of each other, are regulators of the trafficking of AMPA receptor complexes from the endoplasmic reticulum (ER) to the plasma membrane and that both are important determinants for several electrophysiological properties of the receptors ion channel [2,3,4,5, 7, 14, 17, 18]
Summary
The majority of the excitatory synaptic transmission in the mammalian central nervous system is mediated by AMPA4 receptors (AMPARs) These receptors assemble in the endoplasmic reticulum (ER) either as homo- or as heterotetramers [1] of the subunits GluR1 through GluR4. Receptor complex assembly is believed to take place via a two-step mechanism in which interaction of the N-terminal domains leads to the formation of dimers followed by the assembly of such dimers into tetramers [6] This tetramerization is dependent on the amino acid at the Q/R site [7]. Stargazin (␥2) belongs to the family of transmembrane AMPAR regulatory proteins (TARPs), which include the additional members ␥3, ␥4, and ␥8 These proteins are able to rescue the AMPAR-mediated response to glutamate application in cerebellar granule cells of the stargazer mouse and show a distinct expression pattern in the brain [13]. The stargazin-mediated increases in surface expression appear to be entirely independent of the amino acid at the Q/R site, suggesting multiple functionally independent interactions between stargazin and AMPA receptors
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