Divergent roles of a peripheral transmembrane segment in AMPA and NMDA receptors

Abstract

Ionotropic glutamate receptors (iGluRs), including AMPA receptor (AMPAR) and NMDA receptor (NMDAR) subtypes, are ligand-gated ion channels that mediate signaling at the majority of excitatory synapses in the nervous system. The iGluR pore domain is structurally and evolutionarily related to an inverted two-transmembrane K⁺ channel. Peripheral to the pore domain in eukaryotic iGluRs is an additional transmembrane helix, the M4 segment, which interacts with the pore domain of a neighboring subunit. In AMPARs, the integrity of the alignment of a specific face of M4 with the adjacent pore domain is essential for receptor oligomerization. In contrast to AMPARs, NMDARs are obligate heterotetramers composed of two GluN1 and typically two GluN2 subunits. Here, to address the function of the M4 segments in NMDARs, we carry out a tryptophan scan of M4 in GluN1 and GluN2A subunits. Unlike AMPARs, the M4 segments in NMDAR subunits makes only a limited contribution to their biogenesis. However, the M4 segments in both NMDAR subunits are critical for receptor activation, with mutations at some positions, most notably at the extreme extracellular end, completely halting the gating process. Furthermore, although the AMPAR M4 makes a minimal contribution to receptor desensitization, the NMDAR M4 segments have robust and subunit-specific effects on desensitization. These findings reveal that the functional roles of the M4 segments in AMPARs and NMDARs have diverged in the course of their evolution and that the M4 segments in NMDARs may act as a transduction pathway for receptor modulation at synapses.

Figure 1.

Structural features of AMPAR and NMDAR TMDs. (A) Comparison of AMPARs (GluA2, 3KG2, Sobolevsky et al., 2009) and NMDARs (model structure based on 4TLM of GluN1/GluN2B, see Materials and methods) lacking the intracellular CTD. Subunits are colored light orange (GluA2 A and C, GluN1) and gray 60% (GluA2 B and D, GluN2A). For iGluRs, individual subunits as well as the oligomeric complexes are composed of four highly modular domains. Two of these domains are positioned on the extracellular side of the membrane: the amino-terminal domain (ATD) and the LBD. The TMD spans the lipid bilayer and forms the ion channel; in both receptor subtypes at the level of the TMD, the M4 segment of one subunit is associated with the pore domain or ion channel core (M1–M3) of a neighboring subunit (cartoon, right). The fourth domain is the intracellular CTD. (B) View of the TMD for the model NMDAR structure from either the intracellular (bottom-up view) or extracellular (top-down view) side illustrating the association of M4 with an adjacent pore domain. For clarity, one GluN1 and one GluN2A M4 segment are represented as spheres, with VVLGAVE face positions highlighted in red. The center of the pore is indicated by a black dot. (far right) Illustration showing that residues occupying the VVLGAVE face in NMDAR subunits are aligned quite closely, mainly with the M3 segment of an adjacent subunit. The S1-M1 linker of the same subunit is also positioned closely to the extracellular portion of its M4 segments (not depicted). (C) Alignment of the M4 segments and residues on the N- and C-terminal sides for rat AMPAR and NMDAR subunits. Only three residues, a glycine (G), phenylalanine (F), and a glutamate (E), are completely conserved across all subunits (asterisks). Still, residues occupying the VVLGAVE face (boxed) tend to have comparable noncharged (valine [V], leucine [L], methionine [M]) or small (glycine [G], alanine [A], serine [S]) side chains with the exception of a threonine (T) at the VVLGAVE position.

Figure 2.

FSEC to assay AMPAR oligomerization. (A and B) Raw FSEC chromatographs of wild-type GluA2(Q)-EGFP (A) or the same construct containing an alanine substitution at G823 (GluA2(Q)(G823A); B). For quantification, we normalized all chromatographs to the tetramer peak in the wild type for that transfection cycle. For the top panel in each plot, the black line is the original data, the green line the baseline, and the red line is the sum of the individual fits derived from a multi-peak fitting routine (see Materials and methods); the bottom panels show the fraction of the total chromatograph corresponding to, from left to right, tetramer, dimer, or monomer. (C) Mean (±SEM) of the %tetramer (see Materials and methods) for various substitutions of VVLGAVE face positions of the M4 segment in GluA2(Q). Black bars indicate values significantly different from wild type (P < 0.05, ANOVA); asterisks indicate values significantly different from subtle mutations at V813 and V816. Number of independent FSEC runs: wt, 16; V813W, 3; V813A, 4; V813L, 4; V813I, 3; V816A, 4; V816L, 3; V816I, 4; L820A, 4; L820F, 6; G823A, 5; A827S, 3; A827V, 4; V830A, 3; V830L, 3; V830I, 3; E834Q, 6; E834D, 7; E834N, 3. (D) GluA2 M4 segment with VVLGAVE positions shown in red space–filled balls.

Figure 3.

Tryptophan mutagenesis scan of the M4 segments in the GluN1 or GluN2A subunits. (A) Representative whole-cell recordings of current through wild-type GluN1/GluN2A, GluN1(M813W)/GluN2A, or GluN1/GluN2A(S831W). Glutamate (1 mM, shaded box) was applied for 2.5 s. Cells were continuously bathed in glycine (0.1 mM). Holding potential, −70 mV. (B) Mean current amplitudes (±SEM) at −70 mV normalized to current amplitudes for wild-type GluN1/GluN2A (−750 ± 50, n = 18; raw values shown in Table 1). Positions that did not show detectable glutamate-activated currents are demarcated by an “X” (see Materials and methods). Positions that showed current amplitudes significantly less or greater than wild-type are colored blue and green, respectively (P < 0.05, two-tailed Student’s t test, unpaired). The red dots highlight positions homologous to the VVLGAVE face in AMPARs. (C) Orientation of the M3 segments (shadowed), viewed from the center of the pore, to the M4 segments in either AMPARs (left) or NMDARs either GluN2B M3 relative to GluN1 M4 (middle) or GluN1 M3 relative to GluN2B M4 (right). Positions that showed significant changes in current amplitudes are highlighted in blue (reduced), green (greater), or red (no detectable current). For AMPARs, only those positions that showed no detectable current (Salussolia et al., 2011) are indicated, which include the VVLGAVE positions as well as I819.

Figure 4.

Assaying surface expression of NMDARs using pHmystik, a pH-sensitive GFP. (A–D) Fluorescence time trace of GFP intensity as the extracellular bath solution pH was changed from 7.4 to 5.5 (gray bar, 30-s duration) and back again. Shown are HEK293T cells expressing GluN1/pHmystick-GluN2A (A), pHmystik-GluN1/GluN2A(N816W) (B), GluN1(G827)/pHmystick-GluN2A (C), or pHmystik-GluN1/GluN2A(S831W) (D). The representative cell images are from the time-points labeled in panel A; the images were taken at the approximate time points when the baseline fluorescence (F_o), corresponding to image (i), and the test fluorescence (F_test), corresponding to image (ii), were measured as well as an image (iii) after return to pH 7.4. The change in fluorescence (ΔF = F_o − F_test) was used as an index of surface expression. Sampling rate, 5 s. (E) Changes in cell fluorescence (±SEM) at low pH (ΔF, see Materials and methods) for positions in either GluN1 (left) or GluN2A (right). Solid bars indicate values significantly different from their respective wild type either N1/pHmystik-N2A (left) or pHmystik-N1/N2A (right; P < 0.05, t test). The numbers (far right in each plot) indicate the number of cells that showed detectable changes in surface expression relative to the total number of cells tested (see Materials and methods). Only cells that showed detectable changes in fluorescence were included in statistical analysis. (F) Relationship between surface expression, assayed by changes in fluorescence, and whole-cell current amplitudes. For comparison, values (±SEM) are normalized to their respective wild type, either for surface expression (ΔF_norm) or for peak whole-cell current amplitudes (I_{peak norm}; Fig. 3 B). For this ratio of normalized values, a number close to unity implies a correlation between the two parameters, whereas a number much greater than unity indicates surface expression is much greater than expected from whole-cell currents.

Figure 5.

Single-channel recordings of wild-type NMDARs and NMDARs with tryptophan substitutions that showed significantly reduced whole-cell current amplitudes. (A) Example single-channel recordings of GluN1/GluN2A, GluN1(M813W)/GluN2A, or GluN1(E834W)/GluN2A. Recordings were performed in the cell-attached configuration with a pipette potential of 100 mV. Downward deflections reflect inward currents. For each construct, the top half shows a low-resolution example (filtered at 1 kHz), and the bottom half shows a higher-resolution portion of the same record (filtered at 3 kHz). (B) Single-channel current amplitudes (top) and equilibrium open probability (eq. P_o; bottom) shown as mean ± SEM (Table 3). Solid bars indicate values significantly different from wild type (P < 0.05, t test). (C) Approximate number of ion channels in the membrane mediating steady-state whole-cell current amplitudes (Table 4).

Figure 6.

Recovery of function in “pore-dead” constructs. (A) Example whole-cell recordings, displayed as in Fig. 3 A, for NMDARs containing double mutations in the GluN2A subunit, L812M, and a tryptophan substituted at either N816W, V820W, or S831W. Receptors containing single tryptophan substitutions of N816, V820, or S831 showed no detectable glutamate-activated whole-cell currents (Fig. 3 B and Table 1). (B) Mean current amplitudes (±SEM) at −70 mV for single or double mutation constructs. Positions that did not show detectable glutamate-activated currents are demarcated by an “X.” Number of whole-cell current recordings for each construct: GluN1/GluN2A(N816W), 9; GluN1/GluN2A(L812M/N816W), 5; GluN1/GluN2A(V820W), 8; GluN1/GluN2A(L812M/V820W), 4; GluN1/GluN2A(S831W), 9; GluN1/GluN2A(L812M/S831W), 6.

Figure 7.

Impact of M4 substitutions on NMDAR desensitization. (A) Representative whole-cell recordings of current through wild-type GluN1/GluN2A (gray traces) or NMDARs containing a tryptophan substitution in the M4 segment (black traces). Currents were recorded as in Fig. 3. (B) Mean (±SEM) of the %Des or normalized weighted τ for wild-type GluN1/GluN2A or GluN1/GluN2A containing tryptophan substitutions in either the GluN1 (left) or GluN2A (right) M4 segments. Raw values and details of number of recordings made are shown in Table 5. Solid bars indicate values significantly different from wild type (P < 0.01, t test). We used a more stringent level of significance for this analysis to focus on only those positions with prominent effects on desensitization. Dots highlight positions homologous to the VVLGAVE face in AMPARs.

Figure 8.

Substitutions of the VVLGAVE face in GluA2 have no or only weak effects on receptor desensitization. (A) Example outside-out recordings from wild-type GluA2(Q)-EGFP (top trace) or V816I (bottom trace) in response to 100-ms pulses of glutamate. (B) Mean (±SEM) %Des (left) and rate of desensitization (right). Black bars indicate values significantly different from wild type (P < 0.05, t test). Many of the tested positions had strong effects on receptor tetramerization (Fig. 2). We also tested GluA2(L832W), a position on the side of the helix facing away from the pore domain. Number of outside-out patches for each construct: wt, 9; V813I, 4; V816I, 4; L820F, 5; G823A, 4; A827S, 4; V830L, 4; L832W, 4; E834Q, 4; E834D, 4; E834N, 3.

Figure 9.

Differential roles of M4 segments in AMPAR and NMDAR function. (A and B) The M4 segments, viewed from the center of the pore, in either AMPARs (A) or NMDARs (B) either GluN1 M4 (left) or GluN2B M4 (right). The M3 segment from the adjacent subunit (see Fig. 3 C) is hidden to emphasis other M4 interactions, including the S1-M1 LBD-TMD linker from the same subunit and the M1 transmembrane segment from the neighboring subunit. Positions that showed significant changes in current amplitudes are highlighted in blue (reduced), green (greater), or red (no detectable current). For AMPARs, only those positions that showed no detectable current (Salussolia et al., 2011) are indicated, which include the VVLGAVE positions as well as I819. Subunits are colored light orange (GluA2 A and C, GluN1) and gray 60% (GluA2 B and D, GluN2B). The M4 is represented as a cartoon, whereas the S1-M1 and the adjacent M1 are shown as dots.