In principle, for receptors composed of two GluK2 and two GluK3 subunits, two arrangements are possible: (1) pairs of LBD homodimers,

one composed of GluK3 containing two zinc binding sites, with no zinc binding sites in the GluK2 homodimer; and (2) pairs of LBD heterodimers, each containing one zinc binding site formed by residues Q756, D759, CP868596 and H762 in GluK3 and D729 in GluK2 (Figure 8C). To distinguish between these two possibilities, we measured the effect of zinc on receptors composed of GluK2b and GluK3(D730A) in the presence of 1 μM UBP310 to record primarily the activity of heteromeric receptors. Application of zinc (100 μM) led to potentiation of currents (Figures 8D and 8E), similar to WT receptors, whereas for GluK3(D730A) mutant homomeric dimers, zinc potentiation was abolished (Figure 6E). This result is consistent with hypothesis (2). Moreover, in cells transfected with GluK2b(D729A) and GluK3, zinc did not potentiate currents (Figures 8D and 8E), strongly suggesting that the zinc binding site is lost in these heteromeric receptors. Again, this is consistent with hypothesis (2), namely that heteromeric GluK2/GluK3 contains at least an LBD heterodimer and, if composed of two GluK2 and two GluK3 subunits, is arranged as a pair of heterodimers PI3K inhibitor at the level of the LBDs. Our results identify zinc as a positive allosteric

modulator of KARs containing the GluK3 subunit and provide a molecular and mechanistic basis for this allosteric modulation. We identify critical amino acids at the interface between the LBD of two partner subunits that form a pocket

for zinc binding. Zinc stabilizes the interface by cross-bridging the two partner LBDs in the dimer. By its action as a counter ion that reduces repulsion between opposed aspartate side chains, hence strongly reducing desensitization, zinc binding translates into potentiation of the GluK3 response. Our data also provide a mechanistic and structural explanation for the specific properties of the GluK3 subunit of KARs and reveal important information about KAR architecture. In particular, our study provides a structural explanation for the functional differences between the two closely related KAR subunits GluK2 and GluK3 and about the probable arrangement of subunits in a heteromeric GluK2/GluK3 receptor, the only native GluK3-containing most receptor identified so far (Pinheiro et al., 2007). The positive allosteric modulation of KARs by zinc appears as a specific feature of GluK3. Homomeric GluK1 and GluK2, as well as GluK2/GluK4 and GluK2/GluK5, are inhibited by zinc in the concentration range that potentiates GluK3 (this study and Mott et al., 2008). The properties of GluK3, especially the fast desensitization and low agonist sensitivity, set it apart from the other KARs (Perrais et al., 2010; Schiffer et al., 1997). We previously showed that the properties of GluK3 are dominant over those of GluK2 when expressed in heteromeric combinations (Perrais et al.