Modal gating of GluN1/GluN2D NMDA receptors

Abstract

GluN2D-containing NMDA receptors are characterized by an unusually low open probability (0.023), even in the presence of saturating glutamate and glycine. Here, we show that recombinant GluN1/GluN2D NMDA receptors can enter brief periods with exceptionally high open probability (0.65) in excised outside-out and cell-attached single channel recordings. GluN1/GluN2D channels during the enhanced gating mode have similar open durations as occurs outside of the high open probability burst of activity. However, the periods in the high gating mode only exhibit 4 brief closed duration exponential components similar to the briefest observed for openings outside the burst. GluN1/GluN2D receptors also open to a more prominent subconductance level compared to activity outside the high open probability burst. Evaluation of a five-state NMDA receptor gating model suggests that both the opening and closing rate constants differ for the periods of higher open probability compared to the high open probability arm of a gating model previously published for GluN1/GluN2D fit to a representative full length single channel recording. These data demonstrate that GluN2D-containing NMDA receptors can enter a conformation or mode that allows the pore to gate with high probability.

Fig. 1

GluN1/GluN2D receptors exhibit high gating modes in excised outside-out patches. A, A representative trace of an outside-out GluN1-1a/GluN2D single channel recording exhibiting a mode of high open probability (box) than is sustained for 470 ms. The full burst is shown in (B) and expanded further in (C). An open probability stability plot is given in (D), where the P_OPEN of the recording was analyzed in 100 ms increments. Note the sharp increase in P_OPEN at about 90 s into the recording when the channel displays a high gating mode (arrow). E, A representative open-point histogram of fitted channel amplitudes for a high gating mode from the GluN1-1a/GluN2D single channel recording shown in A; this histogram was fitted with two Gaussian components, which we interpret to reflect two levels with chord conductances of 32 pS and 53 pS.

Fig. 2

The high open probability bursts have similar open time components but fewer shut time components than the GluN1/GluN2D single channel recordings for low open probability periods. A, Composite open time histograms from 8 bursts in 6 patches for the high open probability bursts (gray) and the low gating mode in 6 GluN1/GluN2D single channel recordings were best fit by the sum of two exponential components. The high open probability bursts had significantly longer open time components than the openings that occurred outside of the high open probability bursts. B, The composite shut time histogram for all high open probability bursts was best fit by four exponential components, while the histogram for the low open probability periods from the 6 single channel recordings was best fit with 7 exponential recordings. The longer shut time components are absent in the high open probability bursts. There were a total of 1110 open and 1118 closed durations fitted for the high open probability bursts.

Fig. 3

GluN1-1b/GluN2D receptors enter high gating modes in cell-attached patches. A, A representative trace of a cell-attached GluN1-1b/GluN2D receptor single channel recording shows a high gating mode that endures for 260 ms. The entire period of high gating mode is given in (B) and is expanded in (C).

Fig. 4

Gating rates differ during high probability bursts. A, A model with three closed and two open states that previously has been used to describe GluN1/GluN2A and GluN1/GluN2B single channel recordings (Popescu and Auerbach, 2003; Popescu et al., 2004; Erreger et al., 2005; Zhang et al., 2008; Amico-Ruvio and Popescu, 2010) as well as describe each arm of a GluN1/GluN2D model (Vance et al., 2012) was fitted to the composite sequence of single channel openings during the high gating for all recordings during high gating mode. The open probability was considerably higher than that predicted by the upper, fast-gating arm of a model developed for GluN1/GluN2D single channel recordings (Vance et al., 2012) fit to the normal, low mode openings within the representative recording in Fig. 1 (open probability of 0.012). B, The shut time histogram for the composite of all 8 high gating mode bursts is given, overlaid with the probability density function predicted by fitting the gating model to the data. C, The open time histogram for the composite of all 8 high gating mode bursts is given, overlaid with the probability density function predicted by fitting the gating model to the data.