NMDA Receptors Require Multiple Pre-opening Gating Steps for Efficient Synaptic Activity

Abstract

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate fast excitatory synaptic transmission in the nervous system. Applying glutamate to outside-out patches containing a single NMDAR, we find that agonist-bound receptors transition to the open state via two conformations, an "unconstrained pre-active" state that contributes to fast synaptic events and a "constrained pre-active" state that does not. To define how glutamate drives these conformations, we decoupled the ligand-binding domains from specific transmembrane segments for GluN1 and GluN2A. Displacements of the pore-forming M3 segments define the energy of fast opening. However, to enter the unconstrained conformation and contribute to fast signaling, the GluN2 pre-M1 helix must be displaced before the M3 segments move. This pre-M1 displacement is facilitated by the flexibility of the S2-M4 of GluN1 and GluN2A. Thus, outer structures-pre-M1 and S2-M4-work in concert to remove constraints and prime the channel for rapid opening, facilitating fast synaptic transmission.

Figure 1.. Structure and topology of NMDARs

(A) NMDARs consist of four modular domains: extracellular amino-terminal (ATD) and ligand-binding (LBD) domains; the transmembrane domain (TMD) forming the ion channel; and an intracellular C-terminal domain (CTD). Subunits are colored light orange (GluN1) and gray (GluN2B). 6WHR (Chou et al., 2020). (B) Topology of an individual subunit. LBD lobes are colored green (D1) or red (D2). The ion channel is formed by 3 transmembrane segments, M1, M3, and M4, and an intracellular M2 pore loop. Transmembrane segments are colored according to the LBD lobe to which they are connected. (C) Bridging the LBD and TMD in each subunit are three LBD-TMD linkers: S1-M1 (D2-M1), M3-S2 (D2-M3), and S2-M4 (D1-M4). The most membrane proximal secondary structures in the LBD are: β10 (S1-M1); αE (M3-S2); and αK (S2-M4). With agonist binding these structures undergo movements that precede channel opening (Zhu et al., 2016, Tajima et al., 2016, Twomey et al., 2017, Chen et al., 2017a, Chou et al., 2020). Pre-M1 is a short helix in S1-M1 that surrounds the corresponding M3 segment (Sobolevsky et al., 2009).

Figure 2.. Agonist applications to outside-out patches containing a single wild-type GluN1/GluN2A

(A & B) Membrane currents from an outside-out patch (10 consecutive traces from same recording) containing a single wild-type GluN1/GluN2A channel. Traces in (B) are those in (A) but on an expanded time scale. The patch was exposed to pulses (1 s) of glutamate (1 mM) in the continuous presence of glycine (0.1 mM). Current responses reflected either successes (channel opening) or failures (red highlight), with successes showing variations to 1^st opening (blue highlights). Currents were sampled at 50 kHz (displayed at ~ 1 kHz). Holding potential, −70 mV. (C) Success rate (1 - failure rate) for the average of all patches (18 total patches, dots represent individual data points) or the global success rate (1011 successes out of 1279 total trials). (D) Dwell time histogram of the latency to 1^st opening. Histogram was best fit by 2 exponentials (dashed lines). (E) Example summed current. Patch had 140 (123 successes) applications. (F) Bar graph (mean ± SEM) showing activation rates (10–90% rise time) for summed currents (13 patches) from outside-out patches or from currents in the whole-cell (wc) mode (Table S1). Values were not significantly different (p = 0.069, t-test). (G & H) Potential pathways for the transition to the open state. A₄R is the receptor fully bound with agonist, which occurs rapidly after the start of the glutamate application because we used supersaturating concentrations.

Figure 3.. M3-S2 insertions in GluN1 or GluN2A alter the energy for fast opening

(A & B) Overlaid AMPAR structures of αE, M3-S2, and the top of M3 in the closed (5WEK) or open (5WEO) conformations (Twomey et al., 2017). Closed state is light gold (A/C)(~GluN1) (A) or gray (B/D)(~GluN2A) (B). Open state is red (D2-associated). Values shown are the difference between the closed and open states measured from the circle at the mouth of the pore to the last αC in αE (see STAR Methods). (C & D) Dwell time histogram for insertions in GluN1 M3-S2 (G666+1G) (C) or in GluN2A M3-S2 (G664+1G) (D). Histograms were best fit by 2 exponentials (dashed lines). Gray line is wild type. (E) Success rate (1 - failure rate) for the average of all patches for wild type, GluN1 M3-S2 +1G (9 total patches) (global success rate, 0.62; 315 out of 505 trials), or GluN2A M3-S2 +1G (10 total patches) (global success rate, 0.51; 415 out of 808 trials). ***p < 0.001, t-test. (F) Efficiency (=successes × fraction fast component)/total number of trials) of fast component. (G) Energy required for fast component is derived from a log transformation of the forward rate constant for activation (see STAR Methods).

Figure 4.. S1-M1 insertions in GluN1 or GluN2A reduce NMDAR gating

(A & D) Primary amino acid sequences and tertiary structures of the GluN1 (A) and GluN2B (D) S1-M1 linkers and surrounding regions. Gray shading in primary sequence highlight pre-M1 helices which is often not well-defined in GluN2B subunits (Figure S5). Circles indicate number of side chains in the closed state (6WHR) around that position [open, <6 Å; solid, 6–10 Å; red, >10 Å]. Arrows indicate insertions sites, with those in blue indicating few local interactions, whereas those in red indicate more interactions. (B & E) Representative traces from on-cell single-channel patches at −100 mVs. Experiments were for wild-type and constructs containing single insertions either in GluN1 (B) or GluN2A (E) S1-M1. In each panel, upper trace shows 5 seconds of recording (1 kHz filtering), while lower trace is an expanded section (4 kHz). Closed (C) and open (O) states are indicated. (C & F) Single channel eq. P_o, MCT, and MOT (mean ± SEM) for wild-type and S1-M1 insertion constructs either in GluN1 (C) or GluN2A (F) (see Table S3). Dashed lines indicate mean wild-type values. Solid bars indicate values significantly different from wild type (p < 0.05, t-test). ‘X’ indicates no single channel currents could be detected.

Figure 5.. Insertions in S1-M1 linkers slow receptor activation

(A & B) Overlaid AMPAR structures of β10, S1-M1, pre-M1, and the top of M1 (see Figures 3A & 3B for details). (C) Current traces from an outside-out patch of a single NMDAR containing an insertion in GluN2A S1-M1 (G542+1G). Note frequent extended delays to opening (blue). (D & E) Dwell time histograms for GluN1 S1-M1 (I546+1G) (D) (best fit by 2 exponentials, dashed lines) and GluN2A S1-M1 (G642+1G) (E) (single exponential). (F-H) Data analyzed and displayed as in Figures 3E–3G. (F) Success rates for wild type, GluN1 S1-M1+1G (12 total patches) (global success rate, 0.65; 414 out of 639 trials), and GluN2A S1-M1+1G (7 total patches) (global success rate, 0.55; 311 out of 561 trials). *p < 0.05, ***p < 0.001, t-test. (I & J) Glycine applications, in continuous glutamate, to single channel patches. Mean delay to 1st opening (I) and failures (J) (mean ± SEM) for wild-type and insertions in S1-M1s. Solid bars indicate values significantly different from wild type; asterisks indicate those different from corresponding glutamate application (*p < 0.05,*p<0.01, ANOVA, Tukey pairwise). For glycine applications: wild-type, n = 5; GluN1(I546+1G), n = 3; and GluN2A(G542+1G), n = 3.

Figure 6.. Insertions in the GluN1 or GluN2A S2-M4 linkers potentiate gating

(A & D) Primary amino acid sequences from the GluN1 and GluN2A S2-M4 linkers and surrounding regions. (B & E) Representative traces of S2-M4 insertion constructs. (C & F) Single channel eq. P_o, MCT, and MOT (mean ± SEM) for wild-type and S2-M4 insertions either in GluN1 (C) or GluN2A (F) (Table S4).

Figure 7.. NMDARs containing insertions in GluN1 or GluN2A S2-M4 linkers only activate through the fast pathway

(A & B) Overlaid AMPAR structures of αK, S2-M4, and the top of M4, displayed as in Figures 3A & 3B except that the open state is green (D1-associated). (C) Current traces from an outside-out patch of a single NMDAR containing an insertion in the GluN2A S2-M4 (V807+1G). See Figure 2A for details. (D & E) Dwell time histograms for GluN1 S2-M4 (S802+1G) (D) or GluN2A S2-M4 (V807+1G) (E). Histograms best fit by single exponentials. (F-H) Data displayed and analyzed as in Figures 3E–3G. (F) Success rates for wild type, GluN1 S2-M4+1G (10 total patches) (global success rate, 0.99; 304 out of 304 trials), and GluN2A S2-M4+1G (7 total patches) (global success rate, 0.97; 213 out of 219 trials). ***p < 0.001, t-test.

Figure 8.. Outer structures must rearrange for efficient pore opening

Model of NMDAR activation. Movements of the outer structures - GluN1 M4 (gold circle) and GluN2A pre-M1 and M4 (inner gray circles) - regulate entry from agonist-bound closed (A₄R) (left) to either the unconstrained (upper) or constrained (lower) pre-active states. To efficiently enter the unconstrained state, the pre-M1 helices as well as the S2-M4/M4 must be displaced (upper). In contrast, if the GluN2A pre-M1 helix is not displaced, the receptor enters the constrained state, where it still can open but requires more energy to do so (lower). Pre-activation movements are shown in blue, whereas pore opening movements are shown in red.