AMPA receptors mediate the majority of fast synaptic transmission and underlie several forms of synaptic plasticity. AMPARs also have an important role in several neuronal pathologies. Therefore, studying the structure and function of these receptors is important for understanding general mechanisms of synaptic transmission as well as for the development of new therapies. A recent study identified the apparent binding sites for GYKI 53655 (GYKI) and CP-465,022 (CP) at the interface between the glutamate binding core and the transmembrane domains. The emerging role of transmembrane AMPA receptor regulatory proteins (TARPs) in AMPAR function raises the possibility that the antagonism of these receptors is also affected by TARPs such as stargazin. Here we compare the antagonism of the competitive antagonist CNQX and the negative allosteric modulators GYKI, and CP in the absence and presence of stargazin. We found that stargazin decreases the apparent affinity of GluR1 for CNQX, which is most likely explained by a partial agonistic effect of CNQX. In contrast, stargazin increases the affinity for GYKI, and has only a small effect on CP binding. Because inhibition of recently described GYKI insensitive receptors is restored by co-expression with stargazin, our data suggest that the identified residues do not constitute the full GYKI binding site. We could show that the ectodomain of stargazin controls the change in agonist sensitivity. Mutations in the identified binding regions for GYKI and CP dramatically reduced surface expression. Our data provides further evidence that TARPs alter the conformation of pore-forming subunits and thereby affects antagonist interaction.