Gabriela K. Popescu

Modal gating of NMDA receptors and the shape of their synaptic response

N-methyl-D-aspartate receptor (NMDAR) channels mediate the slow component of excitatory potentials at glutamatergic synapses. They have complex kinetic behavior, and much remains to be understood about NMDAR gating mechanisms and the molecular events that shape the synaptic current. Here we show that an individual NMDAR produces at least three stable patterns of activity. For all modes, channels gate by the same mechanism and can occupy either of two open states. The relative stability of the open states differs across modes because of a common perturbation to the NMDAR structure that may be subject to cellular control. Simulations indicate that native NMDAR-mediated synaptic responses arise mainly from the most common mode, and that the slow rise and decay of the current can be attributed to multiple transitions between fully liganded open and closed states rather than to agonist dissociation.

Reaction mechanism determines NMDA receptor response to repetitive stimulation

At central excitatory synapses, N-methyl-d-aspartate (NMDA) receptors, which have a high affinity for glutamate, produce a slowly rising synaptic current in response to a single transmitter pulse and an additional current after a second, closely timed stimulus. Here we show, by examining the kinetics of transmitter binding and channel gating in single-channel currents from recombinant NR1/NR2A receptors, that the synaptic response to trains of impulses is determined by the molecular reaction mechanism of the receptor. The rate constants estimated for the activation reaction predict that, after binding neurotransmitter, receptors hesitate for ∼4 ms in a closed high-affinity conformation before they either proceed towards opening or release neurotransmitter, with about equal probabilities. Because only about half of the initially fully occupied receptors become active, repetitive stimulation elicits currents with distinct waveforms depending on pulse frequency. This high-affinity/low-efficiency activation mechanism might serve as a link between stimulation frequency and the directionality of the ensuing synaptic plasticity.

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF‐2) and FGFR1, association of FGFR1 with splicing factor‐rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up‐regulation of endogenous TH protein or a transfected TH promoter‐luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK‐mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF‐2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co‐transfected HMW FGF‐2 (which is exclusively nuclear) but not by cytoplasmic FGF‐1 or extracellular FGFs. Promoter transactivation by HMWFGF‐2 was accompanied by an up‐regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK‐) but not by IP6 or suramin. The TH promoter was also transactivated by co‐transfected wild‐type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP‐/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF‐2 was mediated through the cAMP‐responsive element (CRE) and was associated with induction of CREB‐ and CBP/P‐300‐containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.

Kinetic basis of partial agonism at NMDA receptors

Activation of ligand-gated channels is initiated by the binding of small molecules at extracellular sites and culminates with the opening of a membrane-embedded pore. To investigate how perturbations at ligand-binding domains influence the gating reaction, we examined current traces recorded from individual NMDA receptors in the presence of several subunit-specific partial agonists. We found that low-efficacy agonists acting at either the glycine-binding or the glutamate-binding NMDA receptor subunits had very similar effects on the receptors activation reaction, possibly reflecting a high degree of coupling between the two subunit types during gating. In addition, we found that partial agonists increased the height of all energy barriers encountered by NMDA receptors during activation. This result stands in sharp contrast to the localized effects that have been observed for pentameric ligand-gated channels and may represent a previously unknown mechanism by which partial agonists reduce receptor activity.

Distinct gating modes determine the biphasic relaxation of NMDA receptor currents

Following brief stimulation, macroscopic NMDA receptor currents decay with biphasic kinetics that is believed to reflect glutamate dissociation and receptor desensitization. We found that the fast and slow decay components arise from the simultaneous deactivation of receptor populations that gate with short and long openings, respectively. Because individual receptors switched infrequently between gating modes, the relaxation time course was largely determined by the proportion of channels in each gating mode at the time of stimulation.

Pregnanolone Sulfate Promotes Desensitization of Activated NMDA Receptors

Neurosteroids are potent neuromodulators which act in part by binding to and modifying the activity of neurotransmitter-gated channels. Pregnanolone sulfate (PAS) is an endogenous neurosteroid that inhibits NMDA receptors and is neuroprotective in vivo. To delineate the mechanism of NMDA receptor inhibition by pregnanolone sulfate we used kinetic analyses of equilibrium single-channel currents recorded from individual GluN1/GluN2A receptors. Results show that PAS (0.1 mm) reduces single-channel open probability by 50% solely by increasing ∼5-fold the mean time spent by receptors in closed conformations. From these data we derive a kinetic scheme that summarizes the effects of PAS on single channel kinetics, accounts for the PAS effects on macroscopic responses and leads us to propose that PAS inhibits NMDA receptor activity by shifting active receptors into desensitized conformations. These findings highlight the neurosteroid inhibitory site on NMDA receptors as a valuable therapeutic target against excitotoxic pathologies including acute and chronic neurodegeneration.

Phosphorylation of Ser1166 on GluN2B by PKA Is Critical to Synaptic NMDA Receptor Function and Ca2+ Signaling in Spines

The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca2+ permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca2+ signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca2+ permeation and Ca2+ signaling in spines. Activation of β-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca2+ permeation, synaptic currents, and Ca2+ rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca2+ signaling at synapses and regulated by the emotional response to stress.

Zinc Effects on NMDA Receptor Gating Kinetics

Zinc accumulates in the synaptic vesicles of certain glutamatergic forebrain neurons and modulates neuronal excitability and synaptic plasticity by multiple poorly understood mechanisms. Zinc directly inhibits NMDA-sensitive glutamate-gated channels by two separate mechanisms: high-affinity binding to N-terminal domains of GluN2A subunits reduces channel open probability, and low-affinity voltage-dependent binding to pore-lining residues blocks the channel. Insight into the high-affinity allosteric effect has been hampered by the receptors complex gating; multiple, sometimes coupled, modulatory mechanisms; and practical difficulties in avoiding transient block by residual Mg(2+). To sidestep these challenges, we examined how nanomolar zinc concentrations changed the gating kinetics of individual block-resistant receptors. We found that block-insensitive channels had lower intrinsic open probabilities but retained high sensitivity to zinc inhibition. Binding of zinc to these receptors resulted in longer closures and shorter openings within bursts of activity but had no effect on interburst intervals. Based on kinetic modeling of these data, we conclude that zinc-bound receptors have higher energy barriers to opening and less stable open states. We tested this model for its ability to predict zinc-dependent changes in macroscopic responses and to infer the impact of nanomolar zinc concentrations on synaptic currents mediated by 2A-type NMDA receptors.

Stationary Gating of GluN1/GluN2B Receptors in Intact Membrane Patches

NMDA receptors are heteromeric glutamate-gated channels composed of GluN1 and GluN2 subunits. Receptor isoforms that differ in their GluN2-subunit type (A-D) are expressed differentially throughout the central nervous system and have distinct kinetic properties in recombinant systems. How specific receptor isoforms contribute to the functions generally attributed to NMDA receptors remains unknown, due in part to the incomplete functional characterization of individual receptor types and unclear molecular composition of native receptors. We examined the stationary gating kinetics of individual rat recombinant GluN1/GluN2B receptors in cell-attached patches of transiently transfected HEK293 cells and used kinetic analyses and modeling to describe the full range of this receptors gating behaviors. We found that, like GluN1/GluN2A receptors, GluN1/GluN2B receptors have three gating modes that are distinguishable by their mean open durations. However, for GluN1/GluN2B receptors, the modes also differed markedly in their mean closed durations and thus generated a broader range of open probabilities. We also found that regardless of gating mode, glutamate dissociation occurred approximately 4-fold more slowly (k(-) = 15 s(-1)) compared to that observed in GluN1/GluN2A receptors. On the basis of these results, we suggest that slow glutamate dissociation and modal gating underlie the long heterogeneous activations of GluN1/GluN2B receptors.

Modes of glutamate receptor gating.

Abstract  The time course of excitatory synaptic currents, the major means of fast communication between neurons of the central nervous system, is encoded in the dynamic behaviour of post‐synaptic glutamate‐activated channels. First‐pass attempts to explain the glutamate‐elicited currents with mathematical models produced reaction mechanisms that included only the most basic functionally defined states: resting vs. liganded, closed vs. open, responsive vs. desensitized. In contrast, single‐molecule observations afforded by the patch‐clamp technique revealed an unanticipated kinetic multiplicity of transitions: from microseconds‐lasting flickers to minutes‐long modes. How these kinetically defined events impact the shape of the synaptic response, how they relate to rearrangements in receptor structure, and whether and how they are physiologically controlled represent currently active research directions. Modal gating, which refers to the slowest, least frequently observed ion‐channel transitions, has been demonstrated for representatives of all ion channel families. However, reaction schemes have been largely confined to the short‐ and medium‐range time scales. For glutamate receptors as well, modal gating has only recently come under rigorous scrutiny. This article reviews the evidence for modal gating of glutamate receptors and the still developing hypotheses about the mechanism(s) by which modal shifts occur and the ways in which they may impact the time course of synaptic transmission.