Lateral hypothalamus hypocretin/orexin glucose-inhibited neurons promote food seeking after calorie restriction.

OBJECTIVE: The present study tests the hypothesis that changes in the glucose sensitivity of lateral hypothalamus (LH) hypocretin/orexin glucose-inhibited (GI) neurons following weight loss leads to glutamate plasticity on ventral tegmental area (VTA) dopamine neurons and drives food seeking behavior. METHODS: C57BL/6J mice were calorie restricted to a 15% body weight loss and maintained at that body weight for 1 week. The glucose sensitivity of LH hypocretin/orexin GI and VTA dopamine neurons was measured using whole cell patch clamp recordings in brain slices. Food seeking behavior was assessed using conditioned place preference (CPP). RESULTS: 1-week maintenance of calorie restricted 15% body weight loss reduced glucose inhibition of hypocretin/orexin GI neurons resulting in increased neuronal activation with reduced glycemia. The effect of decreased glucose on hypocretin/orexin GI neuronal activation was blocked by pertussis toxin (inhibitor of G-protein coupled receptor subunit Gαi/o) and Rp-cAMP (inhibitor of protein kinase A, PKA). This suggests that glucose sensitivity is mediated by the Gαi/o-adenylyl cyclase-cAMP-PKA signaling pathway. The excitatory effect of the hunger hormone, ghrelin, on hcrt/ox neurons was also blocked by Rp-cAMP suggesting that hormonal signals of metabolic status may converge on the glucose sensing pathway. Food restriction and weight loss increased glutamate synaptic strength (indexed by increased AMPA/NMDA receptor current ratio) on VTA dopamine neurons and the motivation to seek food (indexed by CPP). Chemogenetic inhibition of hypocretin/orexin neurons during caloric restriction and weight loss prevented these changes in glutamate plasticity and food seeking behavior. CONCLUSIONS: We hypothesize that this change in the glucose sensitivity of hypocretin/orexin GI neurons may drive, in part, food seeking behavior following caloric restriction.

BTRX-246040 Acts Through the Ventrolateral Periaqueductal Gray to Exert Antidepressant-Relevant Actions in Mice.

BACKGROUND: BTRX-246040, a nociceptin/orphanin FQ peptide receptor antagonist, is being developed for the treatment of depressive patients. However, the underlying mechanism of this potential antidepressant is still largely unclear. Here, we studied the antidepressant-related actions of BTRX-246040 in the ventrolateral periaqueductal gray (vlPAG). METHODS: The tail suspension test, forced swim test, female urine sniffing test, sucrose preference test, and learned helplessness (LH) combined with pharmacological approaches were employed to examine the antidepressant-like effects and drug effects on LH-induced depressive-like behavior in C57BL/6J mice. Electrophysiological recordings in vlPAG neurons were used to study synaptic activity. RESULTS: Intraperitoneal administration of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Systemic BTRX-246040 (10 mg/kg) resulted in an increased frequency and amplitude of miniature excitatory postsynaptic currents (EPSCs) in the vlPAG. Moreover, slice perfusion of BTRX-246040 directly elevated the frequency and amplitude of miniature EPSCs and enhanced the evoked EPSCs in the vlPAG, which were blocked by pretreatment with the nociceptin/orphanin FQ peptide receptor agonist Ro 64-6198. In addition, intra-vlPAG application of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Moreover, intra-vlPAG pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione reversed both systemic and local BTRX-246040-mediated antidepressant-like behavioral effects. Furthermore, both systemic and local BTRX-246040 decreased the LH phenotype and reduced LH-induced depressive-like behavior. CONCLUSIONS: The results suggested that BTRX-246040 may act through the vlPAG to exert antidepressant-relevant actions. The present study provides new insight into a vlPAG-dependent mechanism underlying the antidepressant-like actions of BTRX-246040.

Lateral hypothalamic orexin glucose-inhibited neurons may regulate reward-based feeding by modulating glutamate transmission in the ventral tegmental area.

Glucose inhibits ∼60% of lateral hypothalamic (LH) orexin neurons. Fasting increases the activation of LH orexin glucose-inhibited (GI) neurons in low glucose. Increases in spontaneous glutamate excitatory postsynaptic currents (sEPSCs) onto putative VTA DA neurons in low glucose are orexin dependent (Sheng et al., 2014). VTA DA neurons modulate reward-based feeding. We tested the hypothesis that increased activation of LH orexin-GI neurons in low glucose increases glutamate signaling onto VTA DA neurons and contributes to reward-based feeding in food restricted animals. N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) currents on putative VTA DA neurons were measured using whole cell voltage clamp recording in horizontal brain slices containing the LH and VTA. Decreased glucose increased the NMDA receptor current for at least one hour after returning glucose to basal levels (P < 0.05; N = 8). The increased current was blocked by an orexin 1 receptor antagonist (P < 0.05; N = 5). Low glucose caused a similar persistent enhancement of AMPA receptor currents (P < 0.05; N = 7). An overnight fast increased the AMPA/NMDA receptor current ratio, an in vivo index of glutamate plasticity, on putative VTA DA neurons. Conditioned place preference (CPP) for palatable food was measured during LH dialysis with glucose. CPP score was negatively correlated with increasing LH glucose (P < 0.05; N = 20). These data suggest that increased activation of LH orexin-GI neurons in low glucose after weight loss, leads to enhanced glutamate signaling on VTA DA neurons, increases the drive to eat rewarding food, and may contribute to weight regain.

Oleic Acid in the Ventral Tegmental Area Inhibits Feeding, Food Reward, and Dopamine Tone.

Long-chain fatty acids (FAs) act centrally to decrease food intake and hepatic glucose production and alter hypothalamic neuronal activity in a manner that depends on FA type and cellular transport proteins. However, it is not known whether FAs are sensed by ventral tegmental area (VTA) dopamine (DA) neurons to control food-motivated behavior and DA neurotransmission. We investigated the impact of the monounsaturated FA oleate in the VTA on feeding, locomotion, food reward, and DA neuronal activity and DA neuron expression of FA-handling proteins and FA uptake. A single intra-VTA injection of oleate, but not of the saturated FA palmitate, decreased food intake and increased locomotor activity. Furthermore, intra-VTA oleate blunted the rewarding effects of high-fat/sugar food in an operant task and inhibited DA neuronal firing. Using sorted DA neuron preparations from TH-eGFP mice we found that DA neurons express FA transporter and binding proteins, and are capable of intracellular transport of long-chain FA. Finally, we demonstrate that a transporter blocker attenuates FA uptake into DA neurons and blocks the effects of intra-VTA oleate to decrease food-seeking and DA neuronal activity. Together, these results suggest that DA neurons detect FA and that oleate has actions in the VTA to suppress DA neuronal activity and food seeking following cellular incorporation. These findings highlight the capacity of DA neurons to act as metabolic sensors by responding not only to hormones but also to FA nutrient signals to modulate food-directed behavior.

Direct versus indirect actions of ghrelin on hypothalamic NPY neurons.

OBJECTIVES: Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons. MATERIALS AND METHODS: Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca2+ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus. RESULTS: Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca2+]c, with an EC50 ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP3) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na+]o suppressed activation, suggesting a role for Na+-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons in situ. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca2+]c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and ω-conotoxin, inhibitors of L- and N-type Ca2+ channels, respectively, while Ni2+, mibefradil, and TTA-P2 completely or partially inhibited ghrelin action, implicating T-type Ca2+ channels. Activation was also sensitive to a spider toxin, SNX-482, at concentrations selective for R-type Ca2+ channels. Nanomolar concentrations of GABA markedly inhibited ghrelin-activation of isolated NPY-GFP neurons, consistent with chronic suppression of ghrelin action in vivo. CONCLUSIONS: NPY neurons express all the molecular machinery needed to respond directly to ghrelin. Consistent with recent studies, ghrelin stimulates presynaptic inputs that activate NPY-GFP neurons in situ. Ghrelin can also directly activate a depolarizing conductance. Results with isolated NPY-GFP neurons suggest the ghrelin-activated, depolarizing current is a Na+ conductance with the pharmacologic properties of SUR1/Trpm4 non-selective cation channels. In the isolated neuron model, the opening of SUR1/Trpm4 channels activates T- and SNX482-sensitive R-type voltage dependent Ca2+ channels, which could contribute to NPY neuronal activity in situ.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation.

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.

Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose.

A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus.

Hypothalamic glucose sensing: making ends meet.

The neuroendocrine system governs essential survival and homeostatic functions. For example, growth is needed for development, thermoregulation maintains optimal core temperature in a changing environment, and reproduction ensures species survival. Stress and immune responses enable an organism to overcome external and internal threats while the circadian system regulates arousal and sleep such that vegetative and active functions do not overlap. All of these functions require a significant portion of the body's energy. As the integrator of the neuroendocrine system, the hypothalamus carefully assesses the energy status of the body in order to appropriately partition resources to provide for each system without compromising the others. While doing so the hypothalamus must ensure that adequate glucose levels are preserved for brain function since glucose is the primary fuel of the brain. To this end, the hypothalamus contains specialized glucose sensing neurons which are scattered throughout the nuclei controlling distinct neuroendocrine functions. We hypothesize that these neurons play a key role in enabling the hypothalamus to partition energy to meet these peripheral survival needs without endangering the brain's glucose supply. This review will first describe the varied mechanisms underlying glucose sensing in neurons within discrete hypothalamic nuclei. We will then evaluate the way in which peripheral energy status regulates glucose sensitivity. For example, during energy deficit such as fasting specific hypothalamic glucose sensing neurons become sensitized to decreased glucose. This increases the gain of the information relay when glucose availability is a greater concern for the brain. Finally, changes in glucose sensitivity under pathological conditions (e.g., recurrent insulin-hypoglycemia, diabetes) will be addressed. The overall goal of this review is to place glucose sensing neurons within the context of hypothalamic control of neuroendocrine function.

Metabolic regulation of lateral hypothalamic glucose-inhibited orexin neurons may influence midbrain reward neurocircuitry.

Lateral hypothalamic area (LHA) orexin neurons modulate reward-based feeding by activating ventral tegmental area (VTA) dopamine (DA) neurons. We hypothesize that signals of peripheral energy status influence reward-based feeding by modulating the glucose sensitivity of LHA orexin glucose-inhibited (GI) neurons. This hypothesis was tested using electrophysiological recordings of LHA orexin-GI neurons in brain slices from 4 to 6week old male mice whose orexin neurons express green fluorescent protein (GFP) or putative VTA-DA neurons from C57Bl/6 mice. Low glucose directly activated ~60% of LHA orexin-GFP neurons in both whole cell and cell attached recordings. Leptin indirectly reduced and ghrelin directly enhanced the activation of LHA orexin-GI neurons by glucose decreases from 2.5 to 0.1mM by 53±12% (n=16, P<0.001) and 41±24% (n=8, P<0.05), respectively. GABA or neurotensin receptor blockade prevented leptin's effect on glucose sensitivity. Fasting increased activation of LHA orexin-GI neurons by decreased glucose, as would be predicted by these hormonal effects. We also evaluated putative VTA-DA neurons in a novel horizontal slice preparation containing the LHA and VTA. Decreased glucose increased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs; 125 ± 40%, n=9, P<0.05) and action potentials (n=9; P<0.05) in 45% (9/20) of VTA DA neurons. sEPSCs were completely blocked by AMPA and NMDA glutamate receptor antagonists (CNQX 20 µM, n=4; APV 20µM, n=4; respectively), demonstrating that these sEPSCs were mediated by glutamatergic transmission onto VTA DA neurons. Orexin-1 but not 2 receptor antagonism with SB334867 (10µM; n=9) and TCS-OX2-29 (2µM; n=5), respectively, blocks the effects of decreased glucose on VTA DA neurons. Thus, decreased glucose increases orexin-dependent excitatory glutamate neurotransmission onto VTA DA neurons. These data suggest that the glucose sensitivity of LHA orexin-GI neurons links metabolic state and reward-based feeding.

Mechanism of inhibition of the GluA2 AMPA receptor channel opening: consequences of adding an N-3 methylcarbamoyl group to the diazepine ring of 2,3-benzodiazepine derivatives.

2,3-Benzodiazepine derivatives are AMPA receptor inhibitors, and they are potential drugs for treating some neurological diseases caused by excessive activity of AMPA receptors. Using a laser-pulse photolysis and rapid solution flow techniques, we characterized the mechanism of action of a 2,3-benzodiazepine derivative, termed BDZ-f, by measuring its inhibitory effect on the channel-opening and channel-closing rate constants as well as the whole-cell current amplitude of the homomeric GluA2Q AMPA receptor channels. We also investigated whether BDZ-f competes with GYKI 52466 for binding to the same site on GluA2Q(flip). GYKI 52466 is the prototypic 2,3-benzodiazepine compound, and BDZ-f is the N-3 methylcarbamoyl derivative. We found that BDZ-f is a noncompetitive inhibitor with a slight preference for the closed-channel state of both the flip and the flop variants of GluA2Q. Similar to other 2,3-benzodiazepine compounds that we have previously characterized, BDZ-f inhibits GluA2Q(flip) by forming an initial, loose intermediate that is partially conducting; however, this intermediate rapidly isomerizes into a tighter, fully inhibitory receptor-inhibitor complex. BDZ-f binds to the same noncompetitive site as GYKI 52466 does. Together, our results show that the addition of an N-3 methylcarbamoyl group to the diazepine ring with the azomethine feature (i.e., GYKI 52466) is what makes BDZ-f more potent and more selective toward the closed-channel conformation than the original GYKI 52466. Our results have useful implications for the structure-activity relationship of the 2,3-benzodiazepine series.

Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation.

The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca(2+) influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

The selectivity of conantokin-G for ion channel inhibition of NR2B subunit-containing NMDA receptors is regulated by amino acid residues in the S2 region of NR2B.

The conantokins are short, naturally occurring peptides that inhibit ion flow through N-methyl-d-aspartate receptor (NMDAR) channels. One member of this peptide family, conantokin-G (con-G), shows high selectivity for antagonism of NR2B-containing NMDAR channels, whereas other known conantokins are less selective inhibitors with regard to the nature of the NR2 subunit of the NMDAR complex. In order to define the molecular determinants of NR2B that govern con-G selectivity, we evaluated the ability of con-G to inhibit NMDAR ion channels expressed in human embryonic kidney (HEK)293 cells transfected with NR1, in combination with various NR2A/2B chimeras and point mutants, by electrophysiology using cells voltage-clamped in the whole-cell configuration. We found that a variant of the con-G-insensitive subunit, NR2A, in which the 158 residues comprising the S2 peptide segment (E(657)-I(814)) were replaced by the corresponding S2 region of NR2B (E(658)-I(815)), results in receptors that are highly sensitive to inhibition by con-G. Of the 22 amino acids that are different between the NR2A-S2 and the NR2B-S2 regions, exchange of one of these, M(739) of NR2B for the equivalent K(738) of NR2A, was sufficient to completely import the inhibitory activity of con-G into NR1b/NR2A-containing NMDARs. Some reinforcement of this effect was found by substitution of a second amino acid, K(755) of NR2B for Y(754) of NR2A. The discovery of the molecular determinants of NR2B selectivity with con-G has implications for the design of subunit-selective neurobiological probes and drug therapies, in addition to advancing our understanding of NR2B- versus NR2A-mediated neurological processes.

Non-strict strand orientation of the Ca2+-induced dimerization of a conantokin peptide variant with sequence-shifted gamma-carboxyglutamate residues.

We have previously found a new mode of metal ion-induced helix-helix assembly for the gamma-carboxyglutamate (Gla)-containing, neuroactive conantokin (con) peptides that is independent of the hydrophobic effect. In these unique "metallo-zipper" assemblies of con-G and con-T[K7gamma], interhelical Ca(2+) coordination induces dimer formation with strictly antiparallel chain orientation in conantokin peptides in which Gla residues are positioned at "i, i+4, i+7, i+11" intervals. In order to probe the property of self-assembly in conantokin peptides with an extended Gla network, a con-T variant (con-T-tri) was synthesized that contains five Gla residues spaced at "i, i+4, i+7, i+11, i+14" intervals. Sedimentation equilibrium analyses showed that Ca(2+), but not Mg(2+), was capable of promoting con-T-tri self-assembly. Oxidation and rearrangement assays with Cys-containing con-T-tri variants revealed that the peptide strands in the complex can orient in both parallel and antiparallel forms. Stable parallel and antiparallel dimeric forms of con-T-tri were modeled using disulfide-linked peptides and the biological viability of these species was confirmed by electrophysiology. These findings suggest that small changes within the helix-helix interface of the conantokins can be exploited to achieve desired modes of strand alignment.

N-methyl-D-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein.

The effects of a synthetic apoE peptide, viz., residues 133-149 (apoE[133-149]), a mimetic that comprises the apoE receptor binding domain, on N-methyl-D-aspartate (NMDA)/glycine-induced ion flow through NMDA receptor (NMDAR) channels, have been investigated. The activity of apoE[133-149] was found to depend on the low-density lipoprotein receptor-related protein (LRP). Competition experiments with receptor-associated protein (RAP) and activated alpha(2)-macroglobulin (alpha(2)M*), two proteins that compete for apoE binding to LRP, demonstrate that apoE[133-149] inhibition of NMDAR function is mediated at a locus in LRP that overlaps with the binding sites of RAP and alpha(2)M*. A coreceptor of LRP, cell surface heparin sulfate proteoglycan, did not function in this system. Additional electrophysiology experiments demonstrated that the inhibitory potency of apoE[133-149] was threefold greater for NMDAR-transfected wild-type Chinese hamster ovary (CHO) cells compared with NMDAR-transfected CHO cells deficient in LRP. Studies with truncation and replacement variants of the apoE peptide demonstrated that the NMDAR inhibitory properties of these peptides correlate with their binding affinities for LRP. These novel results indicate that apoE functions as an inhibitor of NMDAR ion channels indirectly via LRP, and are suggestive of a participatory role for LRP in NMDAR-based neuropathies.

Subtype-selective antagonism of N-methyl-D-aspartate receptor ion channels by synthetic conantokin peptides.

Conantokin-G (con-G), conantokin-T (con-T), a truncated conantokin-R (con-R[1-17]), that functions the same as wild-type con-R, and variant sequences of con-T, were chemically synthesized and employed to investigate their selectivities as antagonists of glutamate/glycine-evoked ion currents in human embryonic kidney-293 cells expressing various combinations of NMDA receptor (NMDAR) subunits (NR), viz., NR1a/2A, NR1a/2B, NR1b/2A and NR1b/2B. Con-G did not substantially affect ion flow into NR1a,b/NR2A-transfected cells, but potently inhibited cells expressing NR1a,b/NR2B, showing high NR2B selectivity. Con-T and con-R served as non-selective antagonists of all of four NMDAR subunit combinations. C-terminal truncation variants of the 21-residue con-T were synthesized and examined in this regard. While NMDAR ion channel antagonist activity, and the ability to adopt the Ca(2+)-induced alpha-helical conformation, diminished as a function of shortening the COOH-terminus of con-T, NMDAR subtype selectivity was enhanced in the con-T[1-11], con-T[1-9], and con-T[1-8] variants toward NR2A, NR1b, and NR1b/2A, respectively. Receptor subtype selectivity was also obtained with Met-8 sequence variants of con-T. Con-T[M8A] and con-T[M8Q] displayed selectivity with NR2B-containing subunits, while con-T[M8E] showed enhanced activity toward NR1b-containing NMDAR subtypes. Of those studied, the most highly selective variant was con-T[M8I], which showed maximal NMDAR ion channel antagonism activity toward the NR1a/2A subtype. These studies demonstrate that it is possible to engineer NMDAR subtype antagonist specificity into con-T. Since the subunit composition of the NMDAR varies temporally and spatially in developing brain and in various disease states, conantokins with high subtype selectivities are potentially valuable drugs that may be used at specific stages of disease and in selected regions of the brain.

Helix-helix interactions between homo- and heterodimeric gamma-carboxyglutamate-containing conantokin peptides and their derivatives.

The conantokins are a family of small, naturally occurring gamma-carboxyglutamate (Gla)-rich peptides that specifically antagonize the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptor. One member of this family, conantokin-G (con-G), undergoes Ca(2+)-mediated self-assembly to form an antiparallel helical dimer. Subunit interactions in this complex are incumbent upon intermolecular Ca(2+) bridging of Gla residues spaced at i, i + 4, i + 7, i + 11 intervals within the monomer. Herein, we further probe the molecular determinants governing such helix-helix interactions. Select variants were synthesized to evaluate the contributions of non-Gla residues to conantokin self-association. Con-G dimerization was shown to be exothermic and accompanied by positive heat capacity changes. Using positional Gla variants of conantokin-R (con-R), a non-dimerizing conantokin, i, i + 4, i + 7, i + 11 Gla spacing alone was shown to be insufficient for self-assembly. The Ca(2+)-dependent antiparallel heterodimerization of con-G and con-T(K7 gamma), two peptides that harbor optimal Gla spacing, was established. Last, the effects of covalently constrained con-G dipeptides on NMDA-evoked current in HEK293 cells expressing combinations of NR1a, NR1b, NR2A, and NR2B subunits of the NMDA receptor were investigated. The antiparallel dipeptide was unique in its ability to potentiate current at NR1a/2A receptors and, like monomeric con-G, was inhibitory at NR1a/2B and NR1b/2B combinations. In contrast, the parallel species was completely inactive at all subunit combinations tested. These results suggest that, under physiological Ca(2+) concentrations, equilibrium levels of con-G dimer most likely exist in an antiparallel orientation and exert effects on NMDA receptor activity that differ from the monomer.

Kinetic mechanism of channel opening of the GluRDflip AMPA receptor.

AMPA-type ionotropic glutamate receptors mediate the majority of fast excitatory neurotransmission in the mammalian central nervous system and are essential for brain functions, such as memory and learning. Dysfunction of these receptors has been implicated in a variety of neurological diseases. Using a laser-pulse photolysis technique, we investigated the channel opening mechanism for GluRD(flip) or GluR4(flip) (i.e., the flip isoform of GluRD), an AMPA receptor subunit. The minimal kinetic mechanism for channel opening is consistent with binding of two glutamate molecules per receptor complex. The GluRD(flip) channel opens with a rate constant of (6.83 +/- 0.74) x 10(4) s(-1) and closes with a rate constant of (3.35 +/- 0.17) x 10(3) s(-1). On the basis of these rate constants, the channel opening probability is calculated to be 0.95 +/- 0.12. Furthermore, the shortest rise time (20-80% of the receptor current response to glutamate) is predicted to be 20 micros, which is approximately 8 times shorter than the previous estimate. These findings suggest that the kinetic property of GluRD(flip) is similar to that of GluR2Q(flip), another fast-activating AMPA receptor subunit.