, 2006). Zinc has also been reported to inhibit native and recombinant KARs. selleck kinase inhibitor Zinc inhibition of KARs is subunit dependent, with KARs containing GluK4 or GluK5 subunits being more sensitive, IC50 ∼1–2 μM, than GluK1-GluK2, IC50 ∼70 μM (Mott et al., 2008). Despite the proposed presynaptic function of GluK3-containing KARs at hippocampal mossy fiber synapses, which are highly enriched in vesicular zinc, modulation of these receptors by zinc has not yet been

addressed. In this study, we show that zinc at micromolar concentrations potentiates recombinant GluK3 receptor currents evoked by glutamate. Zinc markedly slows receptor desensitization and increases apparent affinity for glutamate. By analysis of chimeric GluK2/GluK3 KARs and of GluK3 bearing selected point mutations, we mapped the zinc binding domain to the S2 segment of the LBD, in a region forming Erastin the interface between two GluK3 subunits in an LBD dimer assembly. Crystallographic studies for GluK3 LBD complexes with both glutamate

and kainate revealed that zinc ions bind at multiple sites formed by aspartate, histidine, and glutamate residues, which are present in both the upper and lower lobes of the LBD. Based on these crystal structures, a GluK3 LBD dimer model was generated by superposition of GluK3 monomers on previously solved KAR LBD dimers. This identified D730 as the dimer partner component of the binding site underlying zinc potentiation, together with D759 and H762 from the adjacent subunit. Based on these structure-function Adenosine studies and on modeling of

KAR activity, we show that zinc plays a very distinct role in GluK3-KARs by stabilizing the LBD dimer assembly, thereby reducing desensitization. Given the proposed presynaptic localization of GluK3 close to zinc-containing synaptic vesicles, zinc may be an endogenous allosteric modulator for native GluK3-KARs. We tested the effect of zinc on currents activated by fast application of glutamate on lifted HEK293 cells transfected with GluK3 cDNA. Currents evoked by sustained applications (100 ms) of 10 mM glutamate, a concentration close to the EC50 value for GluK3 (Perrais et al., 2009a; Schiffer et al., 1997), were markedly enhanced with preapplication of 100 μM zinc (Figure 1A; 193% ± 38% of control amplitude, n = 17), and this potentiation was rapidly reversible upon removal of zinc. In contrast to GluK3 potentiation, and as previously reported in Xenopus oocytes ( Mott et al., 2008), zinc reversibly inhibited GluK2 currents at all concentrations tested ( Figures 1A and 1D), with an IC50 of 102 ± 11 μM and a Hill coefficient (nH) of 1.1 ± 0.1 (n = 4–9). Because a glutamate concentration of 10 mM is saturating for GluK2 ( Perrais et al., 2009a), this could mask a potentiating effect of zinc. However, currents evoked by 500 μM glutamate, a concentration below the EC50 for GluK2, were also inhibited by 100 μM zinc (48% ± 10%, n = 10; data not shown).