Two-electrode voltage-clamp recordings.

Oocytes were purchased from Rob Weymouth (Xenopus 1, Dexter, MI) and injected with in vitro-transcribed cRNA encoding NMDA receptor subunits. Expression of triheteromeric receptors in oocytes was performed as previously described (Hansen et al., 2014). Briefly, the ratios of injected cRNAs encoding GluN1 and GluN2 subunits were optimized to produce minimal escape currents from diheteromeric receptors (Fig. 1). The injected oocytes were incubated 2–4 days at 19 °C before electrophysiological recordings were performed at room temperature (23 °C) as previously described (Hansen et al., 2014). Unless otherwise stated, the oocytes were voltage-clamped at −40 mV and the extracellular recording solution contained (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl₂, and 0.01 EDTA (pH 7.4 with NaOH). Oocytes expressing GluN2A- or GluN2B-containing NMDA receptors, including triheteromeric GluN1/2B_C1/2D_C2 receptors, were injected with 20 nl of 50 mM BAPTA approximately 10–30 min before recordings to minimize activity-dependent increases of response amplitudes (Williams, 1993). On the day of experiments with triheteromeric receptors, the fraction of total current response mediated by escaped receptors (i.e. escape current) was always determined and subsequent experiments were only performed if this fraction was <10% (Fig. 1).

A) Co-expression of GluN1 with GluN2B_C1 and GluN2D_C2 produces three populations of functional NMDA receptors, but diheteromeric GluN1/2B_C1/2B_C1 and GluN1/2D_C2/2D_C2 are prevented from expressing at the cell surface due to the unmasked dilysine (KKXX) ER retention signals in the intracellular carboxyl-terminal domain. Triheteromeric GluN1/2B_C1/2D_C2 receptors can traffic to the cell surface following heterodimeric coiled-coil formation between C1 and C2 tags that masks the ER retention signals. B) We co-expressed GluN1, GluN2B_C1, and GluN2D_C2 subunits in Xenopus oocytes and measured current responses activated by maximal glutamate plus glycine using two-electrode voltage-clamp electrophysiology. These current responses are, in theory, mediated by triheteromeric NMDA receptors composed of two GluN1, one GluN2B_C1, and one GluN2D_C2 (i.e. GluN1/2B_C1/2D_C2). However, diheteromeric GluN1/2B_C1/2B_C1 and GluN1/2D_C2/2D_C2 receptors may, in practice, escape ER retention and contribute to the measured current responses. To assess the contribution of escaped receptors to the total current response, we used GluN2B_C1, and GluN2D_C2 subunits with mutations in the agonist binding pocket that abolish glutamate binding and render any receptor containing these subunits non-functional (R519K and T691I mutations in GluN2B and R543K and T715I mutations in GluN2D; indicated as RK+TI). Co-expression of GluN1, GluN2B_C1(RK+TI), and GluN2D_C2 therefore only generates functional GluN1/2D_C2/2D_C2 receptors that may escape ER retention and traffic to the cell surface, while co-expression of GluN1, GluN2B_C1, and GluN2D_C2(RK+TI) only generates functional GluN1/2B_C1/2B_C1 receptors. The bar graph summarizes responses from 6 oocytes from one batch of oocytes for each receptor. C) The representative two-electrode voltage-clamp recordings show responses from recombinant NMDA receptors activated by 100 μM glutamate plus 100 μM glycine. D) The efficiency of selective expression of triheteromeric NMDA receptors can be assessed by determining the functional contribution of escaped receptors using the RK+TI mutations. The fractional escape current, calculated as the sum of escape currents divided by the total current, were consistently smaller than 10% for GluN1–1a and GluN1–1b isoforms in combination with GluN2B_C1 and GluN2D_C2 subunits. The data points are for individual batches of oocytes determined as shown in panels B and C.