Fig. 2. Selective expression of triheteromeric GluN1-1a/GluN1-1b/GluN2 NMDA receptors. (A) Linear representation of the polypeptide chain of GluN1-1a and GluN1-1b isoforms with the amino-terminal domain (ATD), the agonist binding domain (ABD) formed by S1 and S2, the transmembrane domain formed by M1, M2, M3, and M4, and the intracellular CTD. Alternative splicing can produce the GluN1-1b isoform that differs from GluN1-1a by the insertion of 21 amino acids encoded by exon 5 (N1 cassette). GluN1-1aC1 and GluN1-1aC2 (1aCX) as well as GluN1-1bC1 and GluN1-1bC2 (1bCX) were generated by fusing engineered C1 and C2 peptide tags to the CTD. (B) Coexpression of GluN2A with 1aC2 and 1bC1 can produce three populations of functional NMDA receptors. Diheteromeric 1aC2/1aC2/2A and 1bC1/1bC1/2A receptors are prevented from trafficking to the cell surface due the presence of unmasked dilysine (KKXX) ER retention signals in the CTD, whereas heterodimeric coiled-coil formation between C1 and C2 tags in triheteromeric 1aC2/1bC1/2A receptors masks the ER retention signals and enable trafficking to the cell surface. (C) Representative two-electrode voltage-clamp recordings of responses from recombinant NMDA receptors expressed in Xenopus oocytes activated by 100 µM glutamate plus 50 µM glycine. Some 1aC2/1aC2/2A and 1bC1/1bC1/2A receptors may escape ER retention, and therefore contribute to the total NMDA receptor-mediated currents. 1aC2 and 1bC1 subunits with mutations in the agonist binding pocket that abolish glycine binding (indicated as FATL) render any receptor containing these subunits nonfunctional. Coexpression of 1aC2, 1bC1-FATL, and GluN2A therefore generates functional receptors that are escaped 1aC2/1aC2/2A receptors, while coexpression of 1aC2-FATL, 1bC1, and GluN2A generates functional receptors that are escaped 1bC1/1bC1/2 receptors. (D) Coexpression of 1aC2 and 1bC1 (as well as GluN2A or GluN2B) produced robust current responses on days 2–4 after cRNA injection, whereas current responses from 1aC2/1aC2/2 and 1bC1/1bC1/2 receptors that may have escaped ER retention remained small compared with the total currents. Data are mean ± S.D., and each bar is from six oocytes. (E) The efficiency of selective expression of NMDA receptors containing two distinct GluN1 isoforms can be assessed by determining the functional contribution of escaped receptors using the FATL mutations. The fractional escape current, calculated as the sum of escape currents divided by the total current, were always smaller than 10% on days 2–4 after cRNA injection for GluN1 isoforms in combination with either GluN2A or GluN2B. Data are mean ± S.D.