Clones corresponding to rat GluK2a(Q),

GluK2b(Q), and GluK3a were described previously (Jaskolski et al., 2004, 2005; Pinheiro et al., 2007). Clones corresponding to GluK2e/3i and GluK3e/2i were described in Perrais et al. (2009a). Site-directed mutagenesis was performed using QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) with specific oligonucleotides corresponding to each mutant (oligonucleotide forward sequences, primed on their respective WT subunits: GluK3(D759G), 5′-GCTGCAGGAGGAGGGCAAGCTTCACATCATGAAGGAG-3′; GluK3(H762A), 5′-GCTGCAGGAGGAGGACAAGCTGGCCATCATGAAGGAGAAGTGGTGGC-3′; GluK3(D730A), 5′-CGGCGGCCTCATCGCTTCCAAGGGCTACGG-3′; GluK3(D730N), 5′-AACTGCAATCTCACCCAGATCGGCGGCCTCATCaATTCCAAGGGCTACGGCATCGGCACGCCCATGG-3′; GluK3(H492Y), 5′-GGCTCCCCTGACCATCACATATGTCCGAGAGAAGGCC-3′; GluK3(H492A), GS-1101 cost 5′-CCCCTGACCATCACCGCGGTCCGAGAGAAGGCC-3′; GluK2(Y490H), 5′-GCTCCACTGGCTATAACCCACGTGCGTGAGAAGGTCATCG-3′; GluK2(G758D), 5′-CAGCTGCAGGAGGAAGACAAGCTTCAC ATGATGAAGGAG-3′; GluK2b(D729A), 5′-ATTGGCGGCCTTATAGCATCCAAAGGCTATGGC-3′). N-terminal deletion versions of GluK2a(Q) and GluK3a(Q) were generated using restriction site addition by PCR before the amino acid sequence GRI at positions 344 and 347, respectively: GluK2 ΔATD: forward 5′-GAATTCCGGCAGAATAACATTT AACAAAACCAATGG-3′,

reverse 5′-CACCAAATGC CTCCCACTATC-3′ primed on GluK2a; GluK3 ΔATD: forward: 5′-GAATTCAGGACGGATTGTTTTCAACAAAACCAGTGGC-3′, reverse 5′-GGCTTAGAGAAGTCAATGGCCTCCTCTCGGACATGGG-3′ primed on GluK3a. The GluK2/3S2 and GluK3/2S2 chimeras were constructed by exchange of the C-terminal domain of one GluK subunit to the other one using the EcoRV restriction site already selleck present on GluK2 in position 532 on the first transmembrane domain and one created by mutagenesis into the same amino acid sequence in position 534 on GluK3 with a forward primer of 5′-CCCCCTGTCCCCAGATATCTGGATGTACGTGC-3′. All DNAs were verified by restriction analysis and sequencing. HEK293 cells (European Collection of Cell Adenylyl cyclase Cultures) were grown and transfected as previously described by Coussen et al. (2005). Cells were cotransfected using FuGENE 6 (Roche) with green fluorescent

protein (GFP) and GluK2a(Q), GluK3a, or mutated forms of these receptors at a cDNA ratio of 1:3. For experiments on heteromeric GluK2/GluK3 receptors, cells were transfected with GFP, GluK2b(Q), and GluK3a at a DNA ratio of 1:1.5:1.5. Cells were used 1–3 days after transfection and replated the day before recording for lifted cells, or 1–2 days before recording for outside-out patches. Brightly fluorescent, isolated cells were selected for recording. Cells were bathed in a solution containing the following: 145 mM NaCl, 2 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES, adjusted to 320 mOsm/l and pH 7.4 with NaOH, at room temperature. Recording pipettes (resistance 3–5 MΩ) were filled with a solution containing the following: 130 mM CsCH3SO3, 2 mM NaCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES, 4 mM Na2ATP, and 0.