We observed the same effect when the whole S2 segment was substituted in GluK3/K2S2 and GluK2/K3S2 chimeric receptors (Figure 5E). Hence, in addition to its role in the zinc action on GluK3, D759 in GluK3 appears as a key residue to explain the specific desensitization properties of GluK3 as compared to GluK1 and GluK2. We searched for additional residues involved in the binding of zinc in the vicinity of D759. This residue is localized in the turn between helices J and K of

the GluK3 LBD (Venskutonytė et al., 2011), three residues downstream of a conserved histidine near the N terminus of helix K. This conserved histidine, together with D759, is a candidate for residues forming part of the zinc binding site for Selleck Androgen Receptor Antagonist GluK3. To test this hypothesis, GluK3 H762 was replaced by an alanine. As expected, the facilitatory effect of zinc was turned to an inhibitory effect in GluK3(H762A) (peak amplitude 45% ± 6%, n = 5; p = 0.014; Figures 6D and 6F), strongly suggesting that H762 participates in the zinc binding site of GluK3 receptors. In addition, the desensitization kinetics of GluK3(H762A) was faster (τdes = 3.9 ± 0.1 ms, n = 5; p = 0.0009; Figure 6G) than for WT GluK3, ruling out an indirect effect of reduced

desensitization on the effect of zinc in this mutant. To obtain further insight into the zinc binding site on GluK3, three independent crystal structures buy LDK378 were solved for the GluK3 LBD in the presence of zinc: two in a complex with glutamate, and one in

a complex with kainate (Table S2). The structure for the six GluK3 protomers in the glutamate complexes was similar to that reported recently by Venskutonytė et al. (2011) but with small variations in the extent of domain closure, from 25.3° to 23.4°, and larger cavity volumes (299 ± 6 Å3, mean ± SD, n = 6) than the value of 274 ± 4 Å3 reported previously, indicating that the LBD of GluK3 is more similar to GluK1 than GluK2, with cavity volumes of 305 and 255 Å3, respectively (Mayer, 2005). For the two protomers in the GluK3 kainate complex (Figure S1), domain closure was 90% and 70% of that induced by glutamate, indicating that the GluK3 LBD can adopt both more closed, and also more open, conformations than observed previously for the GluK3 kainate complex for Cediranib (AZD2171) which a value of 81% was reported by Venskutonytė et al. (2012). In the crystal forms reported here, the GluK3 protomers assemble as two different dimers, both of which diverge from the canonical arrangement found in full-length GluA2 (Sobolevsky et al., 2009). In the P2221 glutamate and P2221 kainate complexes, two GluK3 protomers are arranged head to tail such that helix D of subunit A packs against the N terminus of helix K in subunit B (Figure 7A). Crystallographic symmetry operations generate a second dimer, arranged in a head-to-head assembly but with a >20 Å lateral displacement of the two protomers such that helix J is packed against helix J of its symmetry mate (Figure S1C).