C-terminal domains of N-methyl-D-aspartic acid receptor modulate unitary channel conductance and gating

Abstract

NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.

FIGURE 1.

Schematic representation of NR subunits. A, scaled illustration of major functional domains and their respective relative sizes in N1 and N2 subunits. B, cartoon illustrating membrane topology and overall shape for each subunit. Indicated are truncations used in this study (red arrows) and putative phosphorylation sites (lines). NTD, N-terminal domain; LBD, ligand-binding domain.

FIGURE 2.

NR single-channel activity. Each panel illustrates a continuous 50-s portion of current recorded from a one-channel HEK293 attached patch expressing wild-type receptors (A) and CTD-truncated receptors (B–D). The P_o values indicated in each panel were calculated for the entire file from which the segment was selected. Arrows point to isolated openings that occur only in receptors lacking N2 CTDs.

FIGURE 3.

Current-voltage relationships of single NRs. Amplitudes of single-channel currents recorded from on-cell patches were measured at several applied voltages for receptors lacking the N1 CTD and for wild-type N1/2A and N1/2B. *, significant difference relative to the corresponding wild-type receptor; n = 5 for each condition (p < 0.05).

FIGURE 4.

Open duration distributions of single NR channels. A and B, histograms of open durations detected in the entire records represented in Fig. 2. Overlaid are probability density functions (thick lines) and individual kinetic components (thin lines) calculated by fitting 5C4O models to each data file. Insets give the calculated time constants (τ) and areas (a) for each open component: E_f, E_Low, E_Med, and E_High. C and D, summary bar graphs illustrate the relative changes in the duration of open components. *, statistically significant differences relative to wild type (p < 0.05).

FIGURE 5.

Kinetic analyses of activation events. A and B, histograms of closed durations detected within bursts defined with τ_crit (see “Experimental Procedures” and text). Overlaid are probability density functions (thick lines) and individual kinetic components (thin lines) calculated by fitting 3C4O models to each active file. Insets give the calculated time constants (τ) and areas (a) for each closed component: E₁, E₂, and E₃. C, state models fitted to the events occurring within bursts in each record. The values on the arrows are the calculated averages, rounded to the first significant figure, for the respective rate constant (s⁻¹). *, rates that were significantly faster (red) or slower (blue) relative to the corresponding wild-type receptor (p < 0.05). D, macroscopic responses to 10-ms applications of 1 mm glutamate were simulated with the models in C (left panel, absolute scale) or recorded experimentally from excised outside-out patches (right panel, normalized to peak current).

FIGURE 6.

Kinetic analyses of desensitization events. A, histograms of closed durations for events that occurred outside of τ_crit defined bursts (see “Experimental Procedures” and text) for WT receptors overlaid with the E₄ and E₅ components for WT (black lines) or CTD truncated (colored lines) receptors calculated by fitting 5C4O models to entire data files. Insets give the calculated time constants (τ) and areas (a) for the E₄ and E₅ closed components. B, summary of measured time constants and areas for E₄ and E₅. *, significant differences relative to wild type (p < 0.05). C, macroscopic responses to 5-s applications of 1 mm glutamate were simulated with the full kinetic models in supplemental Fig. S1 (top panel, absolute scale) or recorded experimentally from whole cells (bottom panel, normalized to peak).