, 2007). Therefore, it was important to determine

the effect of zinc on heteromeric GluK2/GluK3 receptors. To test the specific effects of zinc on GluK2/GluK3 heteromers in cells cotransfected with GluK2 S3I-201 and GluK3, we reduced the likeliness of activating homomeric GluK2 or GluK3 subunits as described previously ( Perrais et al., 2009b). First, the GluK2b(Q) splice variant was used because of its reduced expression at the cell surface as a homomer ( Jaskolski et al., 2004). Second, GluK3 homomeric receptors were specifically blocked with 1 μM UBP310 ( Perrais et al., 2009b). In cells cotransfected with GluK2b(Q) and GluK3, application of 1 μM UBP310 inhibited glutamate-activated currents by 55% (n = 6; p < 0.05). The fraction of current resistant to UBP310 was enhanced by zinc (100 μM) to a similar extent (157% ± 7%, n = 18) as for homomeric GluK3 receptors (p = 0.65; Figures 1B and 1C). The small fraction of homomeric GluK2 receptors at the cell surface would, if anything, SB431542 lead to an underestimation of the potentiation of GluK2/GluK3 receptors by zinc. Therefore, these results clearly demonstrate that heteromeric GluK2/GluK3 receptors

are, like GluK3 receptors, potentiated by zinc. The modulation of GluK3 by zinc showed a dose-dependent biphasic effect: increasing the concentration of zinc up to 100 μM potentiated currents (half-maximal effect around 20 μM), and higher concentrations Phenibut progressively inhibited currents (Figure 1D). In order to fit the dose-response

curve with combined potentiation/inhibition Hill equations, we hypothesized that the inhibition of GluK3 by higher concentrations of zinc was similar to that of GluK2 (a notion supported by the effects of point mutations described in Figure 6). This attempt to separate potentiation and inhibition in the GluK3 dose-response curves yielded an EC50 value of 46 ± 17 μM, nH 1.82 ± 0.95, and a maximal potentiation of 475% ± 47%, although the moderate quality of the combined fit suggests that potentiation and inhibition might not be independent processes. Surprisingly, zinc potentiated currents mediated by GluK2/GluK3 at all concentrations tested (Figure 1D), with an EC50 of 477 ± 1638 μM, nH 0.6 ± 0.4, consistent with a reduced number of binding sites on heteromeric receptors, and a maximal potentiation of 286% ± 195% of control, and by contrast to homomeric GluK3 receptors, there was no inhibition for zinc concentrations up to 1 mM. Zinc could affect GluK3-mediated currents in several ways: it could increase single-channel conductance, increase open probability, allow activation of “silent” receptors, or slow down receptor desensitization. It was shown previously that the low glutamate sensitivity of GluK3 receptors was due to fast transitions of glutamate bound receptors to desensitized states (Perrais et al.