Apparent Positive Cooperativity at a Surface cAMP Receptor in Dictyostelium

In the large species of the cellular slime mold Dictyostelium , cell aggregation is regulated by extracellular cAMP. During aggregation, cAMP is released in pulses from cells in the aggregation centers and these rhythmic signals are propagated through the population by a signal relay system. In addition to triggering the relay response, the pulsatile signals also regulate the chemotactic movement of the cells and early cell differentiation. These different cellular responses to exogenous cAMP are thought to be mediated via cAMP receptors, which appear on the cell surface shortly after starvation.
Using a sensitive assay, the equilibrium binding properties of these receptors were analyzed at low cAMP concentrations. As reported earlier, Scatchard plots of cAMP binding to preaggregative amoebae of D. discoideum strain NP187 in the concentration range 2–500 nM were curvilinear suggesting either receptor heterogeneity or negative cooperative interactions. However, at cAMP concentrations below approximately 1.5 nM, the affinity of the receptors was found to decline as a function of decreasing receptor occupancy. This apparent positive cooperativity was observed with binding sites on crude plasma membranes as well as on intact cells, and it occurred at both 0°C and 22°C. Moreover, apparent positive cooperativity was a property of the receptors on all strains of D. discoideum examined and on one strain of D. purpureum . Unlike preaggregative cells, receptors on postaggregative cells often lacked this property.
The lowest concentration of cAMP pulses that can appreciably stimulate membrane differentiation in strain NP187 was found to be 0.15–1.5 nM. Since similar concentrations of exogenous cAMP have been reported to trigger minimal chemotactic and relay responses in D. discoideum , the apparent positive cooperative behavior of the cAMP receptor might function to generate a steep cellular response threshold.     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Neurotransmitter Receptor Trafficking and the Regulation of Synaptic Strength

Modulation of the strength of synapses is thought to be one of the mechanisms that underlies learning and memory and is also likely to be important in processes of neuropathology and drug tolerance. This review focuses on the emerging role of postsynaptic neurotransmitter receptor trafficking as an essential mechanism underlying the dynamic regulation of synaptic strength.     

Sodium Ions Do Not Stabilize the Selectivity Filter of a Potassium Channel

Ion conduction is an essential function for electrical activity in all organisms. The non-selective ion channel NaK was previously shown to adopt two stable conformations of the selectivity filter. Here, we present solid-state NMR measurements of NaK demonstrating a population shift between these conformations induced by changing the ions in the sample while the overall structure of NaK is not affected. We show that two K⁺-selective mutants (NaK2K and NaK2K-Y66F) suffer a complete loss of selectivity filter stability under Na⁺ conditions, but do not collapse into a defined structure. Widespread chemical shift perturbations are seen between the Na⁺ and K⁺ states of the K⁺-selective mutants in the region of the pore helix indicating structural changes. We conclude that the stronger link between the selectivity filter and the pore helix in the K⁺-selective mutants, compared to the non-selective wild-type NaK channel, reduces the ion-dependent conformational flexibility of the selectivity filter.     

Receptor,Ligand and Transducer Contributions to Dopamine D2 Receptor Functional Selectivity

Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor’s signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D₂R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and β-arrestins. D₂R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D₂R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D₂R mutants in which G protein-dependent (^[Gprot]D₂R) and β-arrestin-dependent signaling (^[βarr]D₂R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D₂R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating ^[Gprot]D₂R and ^[βarr]D₂R conferred control of partial agonism at G protein and/or β-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and β-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D₂R functional selectivity and point to means by which novel in vivo therapies could be modeled.     

On the Synthesis of Neurotransmitter Receptor Agonists with Antagonist Potential

The synthesis of a new type of antagonist is described, capable of inactivating neuroreceptors with heretofore unattainable selectivity and permanence. These antagonists are referred to as mazek agonists (i.e. direct, inhibitory agonists) as they have the high receptor affinity and initial receptor-stimulatory effect of direct agonists and are positively coupled to effector systems. However, like direct antagonists, they have a high receptor affinity and the potential to inhibit or prevent receptor stimulation. The synthesis of the present compounds consisted of the covalent attachment of a tethered dye to three different neurotransmitter analogues, resulting in dye-neuropeptide conjugates with a high affinity for the FMRFa receptor. The dye was prepared from azure B (Az), the neurotransmitter was the neuropeptide FMRFamide (FMRFa), and the dye-neuropeptide conjugates synthesized were Az-CFMRFa; Az-CFMRF and Az-CLRFa. In this procedure, the analogues serve as carrier molecules, bound at one end to the receptor and at the other end to the dye, which is thereby brought into close contact with the receptor. The receptor can then be inactivated by singlet oxygen generated by laser irradiation of the photosensitized receptor.     

The Effects of Metal Ions on the Cytotoxicity and Selectivity of a Histidine-Containing Lytic Peptide

The histidine-containing peptide L5C (PAWRHAFHWAWHMLHKAA) is a histidine-rich lytic peptide. Interactions of some divalent metal ions with peptide L5C and their effects on the cell lysis activity of the peptide were studied. The presence of Cu²⁺ caused a secondary structure change (from random coil to α-helix) which resulted in the loss of cell lysis activity in peptide L5C. Binding of Zn²⁺ to peptide L5C also reduced the lytic activity of the peptide but Zn²⁺ did not affect the secondary structure of the peptides. Instead, Zn²⁺ induced peptide L5C aggregation. Unlike Zn²⁺ and Cu²⁺, Mg²⁺ had no significant effect on the activity of peptide L5C. Further experiments revealed that formed ion-peptide L5C complexes were sensitive to pH and dissociated in acidic solutions. Peptide L5C demonstrated improved pH-selectivity in the presence of trace amount of Zn²⁺. This property of histidine-containing lytic peptides can be used to improve their therapeutic effectiveness in the treatment of cancers.     

Quantitative Relationships of Five Putative Neurotransmitter Receptor Sites in Rat Hippocampal Formation

The hippocampus is well suited for studies of the interrelationships of various neurotransmitter systems in the CNS by reason of its simple laminated organization, defined connections, and variety of identified neurotransmitters. We have studied the biochemical and pharmacological properties of five radiolabeled ligand binding sites in a membrane fraction prepared from rat hippocampal formation. These binding sites are thought to identify recognition sites for neurotransmitter receptors. The rank order of ligand binding sites is [³H]muscimol > [³H]quinuclidinyl benzilate > [³Hdihydroergocryptine > [³H]dihydroalprenolol > ¹²⁵I-labeled α-bungarotoxin. All ligands have a single, saturable, high-affinity binding site. Pharmacological characterization of the ligand binding sites indicates properties consistent with the identification of these sites as neurotransmitter receptors.     

Intrinsic Disorder in the T Cell Receptor Creates Cooperativity and Controls ZAP70 Binding

Many immunoreceptors have cytoplasmic domains that are intrinsically disordered (i.e., have high configurational entropy), have multiple sites of posttranslational modification (e.g., tyrosine phosphorylation), and participate in nonlinear signaling pathways (e.g., exhibiting switch-like behavior). Several hypotheses to explain the origin of these nonlinearities fall under the broad hypothesis that modification at one site changes the immunoreceptor’s entropy, which in turn changes further modification dynamics. Here, we use coarse-grain simulation to study three scenarios, all related to the chains that constitute the T cell receptor (TCR). We find that first, if phosphorylation induces local changes in the flexibility of the TCR ζ-chain, this naturally leads to rate enhancements and cooperativity. Second, we find that TCR CD3ɛ can provide a switch by modulating its residence in the plasma membrane. By constraining our model to be consistent with the previous observation that both basic residues and phosphorylation control membrane residence, we find that there is only a moderate rate enhancement of 10% between first and subsequent phosphorylation events. Third, we find that volume constraints do not limit the number of ZAP70s that can bind the TCR but that entropic penalties lead to a 200-fold decrease in binding rate by the seventh ZAP70, potentially explaining the observation that each TCR has around six ZAP70 molecules bound after receptor triggering. In all three scenarios, our results demonstrate that phenomena that change an immunoreceptor chain’s entropy (stiffening, confinement to a membrane, and multiple simultaneous binding) can lead to nonlinearities (rate enhancement, switching, and negative cooperativity) in how the receptor participates in signaling. These polymer-entropy-driven nonlinearities may augment the nonlinearities that arise from, e.g., kinetic proofreading and cluster formation. They also suggest different design strategies for engineered receptors, e.g., whether or not to put signaling modules on one chain or multiple clustered chains.     

Neurotransmitter Receptor Ligand Binding and Enzyme Regional Distribution in the Pigeon Visual System

The relative importance of acetylcholine, dopamine, endogenous opiates, gamma-aminobutyric acid (GABA), glutamate, glycine, noradrenaline, and serotonin as transmitters in the pigeon visual system was estimated by measuring the activity of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and aromatic amino acid decarboxylase (AAD) as well as the binding of dihydroalprenolol, etorphine, kainic acid, muscimol, serotonin, spiroperidol, strychnine, and quinuclidinyl benzilate (QNB) in the tectum opticum, nucleus rotundus, ectostriatum, dorsolateral thalamus, and hyperstriatum (Wulst). As a nonvisual reference structure, the paleostriatal complex was included in the examination. The regional distribution of most of these parameters was very similar to data reported in the mammalian CNS supporting the hypothesis that the avian tectofugal and thalamofugal visual systems are homologous to the mammalian tecto-thalamo-cortical and retino-geniculo-striate pathways, respectively. On the basis of the low values of their parameters, some transmitters can be excluded as significant contributors in a number of structures. As a hypothesis for further investigations, the presence of cholinergic and serotoninergic systems in the Wulst, possibly originating in the dorsolateral thalamus and nucleus raphe, respectively, and of glycinergic and dopaminergic terminals in the paleostriatal complex is proposed.     

Alterations in Neurotransmitter Receptor Binding in Discrete Areas of the Copper-Deficient Rat Brain

Abstract: Neonatal copper deficiency produced alterations in central neurotransmitter receptors that were selective with respect both to brain region and to neurotransmitter receptor type. Both high- and low-affinity dopamine receptor densities in the corpus striatum were significantly lowered, 55% and 29%, respectively, when expressed on a wet weight basis. There was a significant decrease in the level of muscarinic receptors in the striatum whether expressed on the basis of wet weight (50%) or protein (27%). A smaller reduction in muscarinic receptor density was observed in the cortex, whereas there was no effect of copper deficiency in the cerebellum. The treatment did not change β-adrenergic receptor binding in either the cortex or cerebellum. The affinities of the receptors for the ligands was not affected by the low-copper diet. It was previously reported that copper deficiency produces regionally specific decreases in the concentrations of dopamine and norepinephrine. The greatest reduction occurred in the concentration of dopamine in the corpus striatum. The results from both studies suggest that copper deficiency in post-weanling rats may induce a selective morphological lesion.     

Binding of Lanthanum Ions and Ruthenium Red to Synaptosomes and Its Effects on Neurotransmitter Release

A technique for studying the binding of La3+ to synaptosomes in a double-beam spectrophotometer, using murexide as indicator, is described. The binding of La3+ was very rapid and Scatchard plots revealed two components, with KD values of 0.6 and 27 microM in a Na+-free medium (sucrose medium) and 2.3 and 63 microM in an ionic medium containing 135 mM Na+. The binding of the cationic dye ruthenium red (RuR) showed only one site, with a KD of 3.7 microM. La3+ binding was partially inhibited by RuR and vice versa, and La3+ was also capable of partially displacing RuR previously bound to the synaptosomes, particularly in the sucrose medium. The release of labeled gamma-aminobutyric acid (GABA) stimulated by K+ depolarization was inhibited by La3+ concentrations at or above 1 microM, in the ionic medium, whereas in the sucrose medium 2.5 microM or higher La3+ concentrations notably stimulated the spontaneous release of both GABA and glutamic acid. It is concluded that La3+ and RuR share at least one type of binding site, which is probably the high-affinity La3+ site. Since both La3+ and RuR at low concentrations have been shown to block the depolarization-induced Ca2+ entry in synaptosomes, this site might be related to the voltage-dependent Ca2+ entry involved in neurotransmitter release.     

Effects of Postmortem Delay and Temperature on Neurotransmitter Receptor Binding in a Rat Model of the Human Autopsy Process

Abstract: Studies of neurotransmitter and drug receptor alterations in neurodegenerative disorders have contributed to our understanding of the pathophysiology of these conditions. The effect of postmortem delay in freezing tissue after death and prolonged storage of tissue prior to analysis on receptor binding assays are potential artifacts that may limit interpretation of the effects of disease on receptor populations. We used a rat model of the human autopsy process to study the effects of increasing postmortem delay and storage time on N -methylscopolamine (NMS), p -aminoclonidine (PAC), flunitrazepam (FLU), and spiperone binding in a variety of rat brain regions. The rat brains were cooled using a temperature-controlled environment and thermistor probe to follow cooling curves obtained in human brain. Brains were cooled to either room temperature (22°C) or refrigerator temperature (4°C). For three of the four receptors, receptor binding decreased as postmortem delay before freezing increased, particularly in tissue cooled to room temperature. Unlike binding at other receptor sites, FLU binding increased with increasing postmortem delay to freezing. Different effects on K _D and B _max were noted for each ligand studied. No effects of the freezing process itself or storage at -80°C were detectable.     

Opiate Receptor: Multiple Effects of Metal Ions

Abstract: The opiate antagonist [³H]diprenorphine ([³H]dip), a universal ligand at the μ, δ, and k opiate receptor subtypes, was used to study the effects of Ca-II, Cu-II, Mg-II, Mn-II, and Na⁺ on the rat cerebral opiate receptor. Two categories of effects were observed: (a) those on the binding rate constants and (b) those on binding capacity. (a) Sodium ions increased on- and off-rates on [³H]dip with a rather small net change in receptor affinity. The effects of Na⁺ and the divalent ions Ca-II, Mg-II, and Mn-II were antagonistic to each other. Ca-II, Mg-II, and the more effective Mn-II decreased receptor association and dissociation rates, again with minimal changes in the overall binding affinity in washed membrane homogenates. Previous studies using equilibrium binding analysis alone failed to detect changes in [³H]dip binding kinetics caused by these metal ions. In untreated rat brain homogenates, however, Ca-II (and to a lesser extent Mg-II) decreased [³H]dip binding, an effect distinct from that on the binding rate constants in washed membrane homogenates. (b) In untreated, Tris-buffer homogenates not containing external metal ions, a gradual decline in [³H]dip binding was observed. Cu-II or an equivalent endogenous divalent metal ion was identified as a causative factor, and Mn-II partially reversed this effect. Moreover, the addition of Mn-II stabilized the [³H]dip binding sites at very low concentrations of the metal (nM to μM range) that did not change the binding rate constants and that were in the physiological range of Mn-II in rat brain. This unique effect of Mn-II may represent a physiological function in the regulation of the opiate receptor that is not shared by Mg-II and Ca-II. The opposite effects of Cu-II and Mn-II on the in vitro receptor stability may be related to their opposite pharmacological effect in vivo. Finally, multiple changes of the effects of the tested metal ions on [³H]dip binding were observed during in vitro membrane homogenate dilution, centrifugation, and washing. These changes indicate that the opiate receptor complex as it exists in vivo may lose some of its functions and control mechanisms in vitro.     

Synaptic Membrane Freezing Affects Modulatory Sites in Avian Central Nervous System GABAA Receptor

Studies were carried out to determine whether barbiturates and neurosteroids share common recognition sites at the GABA_A receptor complex in avian CNS. To achieve this, differentially prepared fresh and frozen synaptic membranes were used. Both the barbiturate, pentobarbital, and the neurosteroid, 3-hydroxy-5-pregnan-20-one, were able to stimulate GABA binding in both types of membranes. Stimulation differed markedly when both drugs were added jointly to different treated tissue. In frozen membranes drugs acted synergistically and were differentially displaced by picrotoxinin, while in fresh ones, where both compounds were inhibited by the convulsant, this additivity was absent. Post-freezing wash supernatants were collected and used as a source of putative endogenous factors involved in the above mentioned membrane differences. Addition of a high molecular weight fraction from supernatants to frozen synaptic membranes led to an inhibition of barbiturate and neurosteroid potentiation, as well as a loss of their additive effect. Our results indicate that GABA_A receptor modulation by barbiturates and neurosteroids is affected by synaptic membrane treatment, with a common modulatory site in fresh membranes and separate recognition sites after a freeze-thawing procedure. There may also be endogenous factors involved in overlapping of modulatory sites, which would thus regulate GABA_A receptor functionality by direct interaction with the complex.     

LRRK2 Affects Vesicle Trafficking,Neurotransmitter Extracellular Level and Membrane Receptor Localization

The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2^G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2^G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.     

Potassium Ions and the Inhibitory Process in the Crayfish Stretch Receptor

The effect of the absence of potassium in the bathing solution on the synaptic inhibitory potentials of the crayfish stretch receptor has been studied. The inhibitory potentials were increased in size, i.e. became more hyperpolarizing, in the absence of potassium. Since the resting potential of the cell is increased in the absence of potassium, the alteration of the inhibitory potentials implies that the potassium conductance of the membrane is increased. While other ions, e.g. Cl^-, may also be involved, it seems that the membrane potential during inhibition is mainly dominated by K⁺.     

Heterotropic Cooperativity within and between Protomers of an Oligomeric M(2) Muscarinic Receptor

At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand.