Videos by Gabriela K Popescu
This workshop helps junior investigators consider the challenges and rewards of developing a succ... more This workshop helps junior investigators consider the challenges and rewards of developing a successful research team.
Topics addressed include:
- understanding the overlapping roles of the lab director as employer, mentor and scientist;
- articulating and communicating a research mission
- hiring (and firing) for research and training success;
- understanding the natural process of team assembly, maturation, and change;
- considering legislative, administrative, and cultural aspects of the environment in which the team operates.
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Papers by Gabriela K Popescu
N-Methyl-D-aspartic (NMDA) receptors are excitatory glutamate-gated ion channels. Their activatio... more N-Methyl-D-aspartic (NMDA) receptors are excitatory glutamate-gated ion channels. Their activation is essential for the normal development, maintenance, and plasticity of excitatory synapses in the central nervous system. They function as glutamate-gated Ca2+-permeable channels, require glycine as co-agonist, and can be modulated by myriad of diffusible ligands and cellular cues, including mechanical stimuli. Previously, we found that in cultured astrocytes, shear stress initiates NMDA receptor-mediated Ca2+ entry in the absence of added agonists, suggesting that in addition to being mechanosensitive, NMDA receptors may be mechanically activated. Here, we used controlled expression of recombinant receptors and non-invasive on-cell single-channel current recordings to show that gentle membrane stretch can substitute for the neurotransmitter glutamate in gating NMDA receptor currents. Notably, stretch-activated currents preserved the hallmark features of the glutamate-gated currents, ...
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ABSTRACTTo investigate the role of the N-terminal domains (NTDs) in NMDA receptor signaling we us... more ABSTRACTTo investigate the role of the N-terminal domains (NTDs) in NMDA receptor signaling we used kinetic analyses of one-channel currents and compared the reaction mechanism of recombinant wild-type GluN1/GluN2A and GluN1/GluN2B receptors with those observed for NDT-lacking receptors. We found that truncated receptors maintained the fundamental gating mechanism characteristic of NMDA receptors, which includes a multi-state activation sequence, desensitization steps, and mode transitions. This result establishes that none of the functionally-defined NMDA receptor activation events require the NTD. Notably, receptors that lacked the entire NTD layer retained isoform-specific kinetics. Together with previous reports, these results demonstrate that the entire gating machinery of NMDA receptors resides within a core domain that contains the ligand-binding and the channel-forming transmembrane domains, whereas the NTD and C-terminal layers serve modulatory functions, exclusively.
Biophysical Journal, 2019
N-Methyl-d-aspartate (NMDA) receptors are Ca 2þ -permeable channels gated by glutamate and glycin... more N-Methyl-d-aspartate (NMDA) receptors are Ca 2þ -permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca 2þ -dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca 2þ , we observed robust CDI of whole-cell GluN2A currents (0.42 5 0.05) but no CDI in GluN2B currents (0.08 5 0.07). In contrast, when Ca 2þ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 5 0.03 and 0.67 5 0.02, respectively). To examine how the source of Ca 2þ affects CDI, we recorded one-channel Na þ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca 2þ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (P o ). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high P o GluN2B receptors (GluN1 A652Y /GluN2B). Together, these results show that Ca 2þ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel P o . These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca 2þ load during high P o activation.
The Journal of Neuroscience, 2019
In the CNS, NMDA receptors generate large and highly regulated Ca 2ϩ signals, which are critical ... more In the CNS, NMDA receptors generate large and highly regulated Ca 2ϩ signals, which are critical for synaptic development and plasticity. They are highly clustered at postsynaptic sites and along dendritic arbors, but whether this spatial arrangement affects their output is unknown. Synaptic NMDA receptor currents are subject to Ca 2ϩ -dependent inactivation (CDI), a type of activity-dependent inhibition that requires intracellular Ca 2ϩ and calmodulin (CaM). We asked whether Ca 2ϩ influx through a single NMDA receptor influences the activity of nearby NMDA receptors, as a possible coupling mechanism. Using cell-attached unitary current recordings from GluN1-2a/ GluN2A receptors expressed in human HEK293 cells and from NMDA receptors native to hippocampal neurons from male and female rats, we recorded unitary currents from multichannel patches and used a coupled Markov model to determine the extent of signal coupling (). In the absence of extracellular Ca 2ϩ , we observed no cooperativity ( Ͻ 0.1), whereas in 1.8 mM external Ca 2ϩ , both recombinant and native channels showed substantial negative cooperativity ( ϭ 0.27). Intracellular Ca 2ϩ chelation or overexpression of a Ca 2ϩ -insensitive CaM mutant, reduced coupling, which is consistent with CDI representing the coupling mechanism. In contrast, cooperativity increased substantially ( ϭ 0.68) when overexpressing the postsynaptic scaffolding protein PSD-95, which increased receptor clustering. Together, these new results demonstrate that NMDA receptor currents are negatively coupled through CDI, and the degree of coupling can be tuned by the distance between receptors. Therefore, channel clustering can influence the activity-dependent reduction in NMDA receptor currents.
Biophysical Journal, 2015
Biophysical Journal, 2017
receptors are glutamate-and glycine-gated channels that flux Na þ and Ca 2þ into postsynaptic neu... more receptors are glutamate-and glycine-gated channels that flux Na þ and Ca 2þ into postsynaptic neurons during synaptic transmission. The resulting intracellular Ca 2þ transient is essential to physiological and pathological processes related to synaptic development, plasticity, and apoptosis. It also engages calmodulin (CaM) to reduce subsequent NMDA receptor activity in a process known as Ca 2þ -dependent inactivation (CDI). Here, we used whole-cell electrophysiology to measure CDI and computational modeling to dissect the sequence of events that underlies it. With these approaches, we estimate that CaM senses NMDA receptor Ca 2þ influx at $9 nm from the channel pore. Further, when we controlled the frequency of Ca 2þ influx through individual channels, we found that a kinetic model where apoCaM associates with channels before their activation best predicts the measured CDI. These results provide, to our knowledge, novel functional evidence for CaM preassociation to NMDA receptors in living cells. This particular mechanism for autoinhibitory feedback reveals strategies and challenges for Ca 2þ regulation in neurons during physiological synaptic activity and disease.
Scientific reports, Jan 3, 2017
While studying the physiological response of primary rat astrocytes to fluid shear stress in a mo... more While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca(2+) entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca(2+) influx. Extracellular Mg(2+) at 2 mM did not significantly affect the shear induced Ca(2+) influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.
J Neurochem, 2002
The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular gro... more 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.
The Journal of Biological Chemistry, Nov 24, 1995
A retinol dehydrogenase, RoDH(I), which recognizes holo-cellular retinol-binding protein (CRBP) a... more A retinol dehydrogenase, RoDH(I), which recognizes holo-cellular retinol-binding protein (CRBP) as substrate, has been cloned, expressed, and identified as a short-chain dehydrogenase/reductase (Chai, X., Boerman, M. H. E. M., Zhai, Y., and Napoli, J. L. (1995) J. Biol. Chem. 270, 3900 -3904). This work reports the cloning and expression of a cDNA encoding a RoDH isozyme, RoDH(II). The predicted amino acid sequence verifies RoDH(II) as a short-chain dehydrogenase/reductase, 82% identical with RoDH(I). RoDH(II) recognized the physiological form of retinol as substrate, CRBP, with a K m of 2 mM. Similar to microsomal RoDH and RoDH(I), RoDH(II) had higher activity with NADP rather than NAD, was stimulated by ethanol and phosphatidyl choline, was not inhibited by the medium-chain alcohol dehydrogenase inhibitor 4-methylpyrazole, but was inhibited by phenylarsine oxide and the short-chain dehydrogenase/reductase inhibitor carbenoxolone. Northern blot analysis detected RoDH(I) and RoDH(II) mRNA only in rat liver, but RNase protection assays revealed RoDH(I) and RoDH(II) mRNA in kidney, lung, testis, and brain. These data indicate that short-chain dehydrogenases/reductase isozymes expressed tissuedistinctively catalyze the first step of retinoic acid biogenesis from the physiologically most abundant substrate, CRBP.
The mechanism by which ligand binding at extracellular receptor domains gates a transmembrane ion... more The mechanism by which ligand binding at extracellular receptor domains gates a transmembrane ion-conducting pore is insufficiently understood. Examining a channel's activation reaction in the presence of agonists with distinct efficacies may inform this issue and may help identify agonistdependent transitions. We have recently applied this approach to NMDA receptors composed of GluN1 and GluN2A subunits. Based on our results with several subunit-specific partial agonists we concluded that agonist effects were distributed over several of the multiple transitions that make up NMDA receptor gating and that these changes were subunit independent. Here we examine an additional GluN2A partial agonist, 4-fluoro-D, L-glutamic acid, and we summarize the observed kinetic changes of all nine partial agonists investigated. These results support the premise that regardless of the subunit-type to which they bind, agonists influence multiple equilibria within the NMDA receptor reaction and may stabilize a slightly different family of conformers.
The Journal of general physiology, 2015
N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation re... more N-methyl-d-aspartate (NMDA) receptors are the only neurotransmitter receptors whose activation requires two distinct agonists. Heterotetramers of two GluN1 and two GluN2 subunits, NMDA receptors are broadly distributed in the central nervous system, where they mediate excitatory currents in response to synaptic glutamate release. Pore opening depends on the concurrent presence of glycine, which modulates the amplitude and time course of the glutamate-elicited response. Gating schemes for fully glutamate- and glycine-bound NMDA receptors have been described in sufficient detail to bridge the gap between microscopic and macroscopic receptor behaviors; for several receptor isoforms, these schemes include glutamate-binding steps. We examined currents recorded from cell-attached patches containing one GluN1/GluN2A receptor in the presence of several glycine-site agonists and used kinetic modeling of these data to develop reaction schemes that include explicit glycine-binding steps. Based...
Biophysical Journal, 2015
Biophysical Journal, 2015
Journal of neurochemistry, 2015
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 14, 2015
NMDA receptors mediate excitatory neurotransmission in brain and spinal cord and play a pivotal r... more NMDA receptors mediate excitatory neurotransmission in brain and spinal cord and play a pivotal role in the neurological disease state of chronic pain, which is caused by central sensitization. Bupivacaine is the indicated local anesthetic in caudal, epidural, and spinal anesthesia and is widely used clinically to manage acute and chronic pain. In addition to blocking Na(+) channels, bupivacaine affects the activity of many other channels, including NMDA receptors. Importantly, bupivacaine inhibits NMDA receptor-mediated synaptic transmission in the dorsal horn of the spinal cord, an area critically involved in central sensitization. We used recombinant NMDA receptors expressed in HEK293 cells and found that increasing concentrations of bupivacaine decreased channel open probability in GluN2 subunit- and pH-independent manner by increasing the mean duration of closures and decreasing the mean duration of openings. Using kinetic modeling of one-channel currents, we attributed the obs...
The Journal of general physiology, 2015
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Videos by Gabriela K Popescu
Topics addressed include:
- understanding the overlapping roles of the lab director as employer, mentor and scientist;
- articulating and communicating a research mission
- hiring (and firing) for research and training success;
- understanding the natural process of team assembly, maturation, and change;
- considering legislative, administrative, and cultural aspects of the environment in which the team operates.
Feedback appreciated!
Papers by Gabriela K Popescu
Topics addressed include:
- understanding the overlapping roles of the lab director as employer, mentor and scientist;
- articulating and communicating a research mission
- hiring (and firing) for research and training success;
- understanding the natural process of team assembly, maturation, and change;
- considering legislative, administrative, and cultural aspects of the environment in which the team operates.
Feedback appreciated!