Modal gating of GluN1/GluN2D NMDA receptors

Highlights

• We evaluated GluN2D single channel recordings activated by glutamate and glycine.

• GluN1/GluN2D exhibits modal gating with brief periods of high open probability.

• The high open probability bursts have fewer shut duration components.

• The bursts were best fitted by a 3-closed and 2-open state gating model.

• The high mode opening and closing rate constants differed from the low mode rates.

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.

Introduction

N-methyl-d-aspartate (NMDA) receptors are ionotropic glutamate receptors typically composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Four GluN2 subunits (GluN2A-D) have been identified and are thought to control many of the kinetic properties of the NMDA receptor, including open probability, conductance levels, and deactivation time course (Monyer et al., 1994; Kuner and Schoepfer, 1996; Vicini et al., 1998; Qian et al., 2005; Dravid et al., 2008; Yuan et al., 2009; Traynelis et al., 2010; Vance et al., 2012). In addition, eight GluN1 splice variants, which are formed by mRNA splicing of a single gene, have been identified (Hollmann et al., 1993). The single channel and macroscopic current properties as well as the neuronal expression of GluN2D-containing NMDA receptors differ substantially from other NMDA receptor subunits. Individual recombinant GluN1-1a/GluN2D NMDA receptors have a particularly low single channel open probability of approximately 0.02, which is 25-fold lower than GluN1/GluN2A receptors (open probability around 0.5) (Popescu and Auerbach, 2003; Erreger et al., 2005; Yuan et al., 2009; Vance et al., 2012). GluN2D-containing receptors also exhibit a prominent single channel subconductance level, reduced sensitivity to inhibition by Mg²⁺, and an exceptionally slow deactivation time course following the removal of glutamate (Wyllie et al., 1996; Vicini et al., 1998; Wyllie et al., 1998; Clarke and Johnson, 2006; Yuan et al., 2009; Vance et al., 2011). The expression of the GluN2D subunit peaks early in development and later can only be found in particular regions of the adult brain (Monyer et al., 1994; Standaert et al., 1994; Dunah et al., 1996; Wenzel et al., 1996), including the subthalamic nucleus, substantia nigra, spinal cord, cerebellar Golgi and Purkinje cells, and interneurons (Laurie and Seeburg, 1994; Monyer et al., 1994; Standaert et al., 1994, 1996; Dunah et al., 1996; Wenzel et al., 1996; Goebel and Poosch, 1999; Standaert et al., 1999).

NMDA receptors have long been known to exhibit periods of high open probability. Jahr and Stevens (1987) and Gibb and Colquhoun (1991) reported brief periods of extremely high open probability in single channel recordings of hippocampal NMDA receptors activated by the selective agonist NMDA (Jahr and Stevens, 1987; Gibb and Colquhoun, 1991), while Howe et al., 1988, Howe et al., 1991 identified high open probability bursts in native NMDA receptors in cerebellar granule cells when activated by several agonists, including NMDA, glutamate, and aspartate (Howe et al., 1988, 1991). These high open probability bursts were correlated to an increase in mean open time (Jahr and Stevens, 1987) and were independent of agonist concentration (Gibb and Colquhoun, 1991). More recently, recombinant AMPA and NMDA receptors have been shown to exhibit multiple modes in gating when expressed in HEK 293 cells. Both GluN1/GluN2A (Popescu and Auerbach, 2003; Popescu et al., 2004; Zhang et al., 2008; Kussius and Popescu, 2009) and GluN1/GluN2B (Amico-Ruvio and Popescu, 2010) NMDA receptors exhibit modal gating, although on a much longer time scale than observed in the hippocampal or cerebellar NMDA receptor recordings. Moreover, unlike hippocampal NMDA channels, recombinant GluN2A-containing receptors are capable of entering high-, medium-, and low-gating modes. Modal gating described for GluN1/GluN2A and GluN1/GluN2B receptors also is correlated to mean open time, as open time is longest in high mode and shortest in low mode (Popescu and Auerbach, 2003; Popescu et al., 2004; Zhang et al., 2008; Kussius and Popescu, 2009; Amico-Ruvio and Popescu, 2010; Popescu, 2012). Additionally, single channel recordings of GluA1-4 AMPA receptors also reveal distinct gating modes when recorded in the presence of cyclothiazide to reduce desensitization (Poon et al., 2010; Prieto and Wollmuth, 2010; Poon et al., 2011). While both NMDA and AMPA receptors appear able to enter multiple gating modes, the mechanisms that control modal gating remain elusive.

The goal of this study was to determine whether GluN1/GluN2D NMDA receptors also can undergo modal gating. We show here that GluN1-1a/GluN2D NMDA receptors are capable of entering brief periods of exceptionally high open probability when activated by saturating concentrations of glutamate and glycine in excised, outside-out patch recordings with a single active channel. We further demonstrate that GluN2D-containing receptors can enter a high gating mode regardless of recording condition or the GluN1 splice variant assembled within the receptor, as cell-attached single channel recordings of GluN1-1b/GluN2D receptors also exhibit a high gating mode. Finally, we show that the rate constants of a five-state NMDA receptor gating model are altered when fit to the high open probability bursts from GluN1-1a/GluN2D receptors in outside-out patches that contain a single active channel when compared to the channel in lower open probability gating mode.