Gating reaction mechanism of neuronal NMDA receptors

Abstract

The activation mechanisms of recombinant N-methyl-d-aspartate receptors (NRs) have been established in sufficient detail to account for their single channel and macroscopic responses; however, the reaction mechanism of native NRs remains uncertain due to indetermination of the isoforms expressed and possible neuron-specific factors. To delineate the activation mechanism of native NRs, we examined the kinetic properties of currents generated by individual channels located at the soma of cultured rat neurons. Cells were dissociated from the embryonic cerebral cortex or hippocampus, and on-cell single channel recordings were done between 4 and 50 days in vitro (DIV). We observed two types of kinetics that correlated with the age of the culture. When we segregated recordings by culture age, we found that receptors recorded from early (4-33 DIV) and late (25-50 DIV) cultures had smaller unitary conductances but had kinetic profiles that matched closely those of recombinant 2B- or 2A-containing receptors, respectively. In addition, we examined the effects of cotransfection with postsynaptic density protein 95 or neuropilin tolloid-like protein 1 on recombinant receptors expressed in human embryonic kidney-293 cells. Our results add support to the view that neuronal cultures recapitulate the developmental patterns of receptor expression observed in the intact animal and demonstrate that the activation mechanism of somatic neuronal NRs is similar to that described for recombinant receptors of defined subunit composition.

Fig. 1.

N-methyl-d-aspartate receptor (NR) unitary conductance. A: cell-attached patch-clamp recording configuration used in this study (top) and representative single channel current trace recorded from a native receptor [29 days in vitro (DIV); bottom]. HEK-293 cell, human embryonic kidney-293 cell; V_R, recorded voltage. B: event amplitude histogram for the entire record illustrated in A (21,507 events, 14 min) overlaid with the probability density function calculated for a two-state model. Values are means ± SD. Components are in black [closed (C), 0.01 ± 1.56 pA] and red [open (O), 8.9 ± 1.9 pA]. C: summary of unitary conductance results showing the means (boxes) and ranges (whiskers) for the three conditions indicated.

Fig. 2.

Single channel properties of native NRs. A: NR activities recorded from dissociated neurons segregate into two open probability (P_O) ranges, which correlated roughly with culture age: early neurons (ENs; 4–33 DIV, n = 23) and late neurons (LNs; 25–50 DIV, n = 11). B: Scatterplot of single channel kinetic parameters for native (EN and LN) and recombinant (N1/2B and N1/2A) receptors. C: representative single channel current traces recorded from one EN receptor (top; 11 DIV) and one LN receptor (bottom; 26 DIV). D: histograms of closed (left) and open (right) event durations for the records illustrated in C (EN: green and LN: blue) overlaid with those measured in records from one recombinant N1/2B receptor (top) or N1/2A receptor (bottom; black). Overall probability distributions (thick solid lines) and individual closed (E₁–E₅) and open [fast (O_f), low (O_L), medium (O_M), and high (O_H)] kinetic components (thin solid lines) are also shown.

Fig. 3.

NR gating mechanisms. Rates (in s⁻¹) are given for each transition as the rounded average across all records in each set. Differences were not significant between EN and N1/2B receptors or between LN and N1/2A receptors for any of the transitions estimated (P > 0.05 by Student's t-test).

Fig. 4.

Gating modes of native NRs. Three separate 1-s periods were selected from one EN recording (A) and one LN recording (B). In A and B, traces differ by their mean open durations [mean open time (MOT)] and illustrate activity in one of the low (L), medium (M), and high (H) gating regimes adopted randomly by NRs.

Fig. 5.

Effects of postsynaptic density protein (PSD)95 and neuropilin tolloid-like protein (Neto)1 on macroscopic responses of recombinant NRs. A: whole cell currents were elicited with 5-s applications of 1 mM glutamate from HEK-293 cells transfected with N1 and 2B (top) or with N1 and 2A (bottom) and in combination with PSD95 (left) or Neto1 (right). B: summary of changes in macroscopic desensitization kinetics observed in HEK-293 cells cotransfected NR subunits alone (black) and in conjunction with PSD95 (red) or Neto1 (blue). I_ss/I_pk, steady-state (equilibrium) current-to-peak current; τ_D, time constant of macroscopic desensitization. *P < 0.05 relative to NR subunits alone (by Student's t-test).

Fig. 6.

Effects of PSD95 and Neto1 on recombinant NR single channel kinetic parameters. Statistical graphs show the means (box), medians (line), and ranges (whiskers) of values measured for N1/2B (black), N1/2B + PSD95 (green), N1/2A (gray), N1/2A + PSD95 (red), and N1/2A + Neto1 (orange). MCT, mean closed time. *Significant differences in the variance of these parameters observed in cells transfected with NR subunits alone relative to those observed in cells cotransfected with PSD95 (P < 0.05 by F-test).

Fig. 7.

Effects of PSD95 on recombinant NR kinetics. A and B: summary of changes in closed component durations of single channel records obtained from cells expressing N1/2B (A) or N1/2A (B) receptors alone [PSD95(−), with mean durations below] and in conjunction with PSD95 [PSDD95(+)] as well as the reaction mechanism deduced from single channel records. All rates are in s⁻¹; mean rate constants for NRs alone are shown in Fig. 3. *Significant differences in means relative to cells expressing NRs alone (P < 0.05 by Student's t-test). Macroscopic traces were simulated with the models shown in Fig. 3 (black) and overlaid with traces simulated with the models given at the left (gray) for each receptor subtype.