Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

Liaw, Shwu-Jen; Kuo, I-Chun; Eisenberg, David

doi:10.1002/pro.5560041114

Cited by 94 publications

(103 citation statements)

References 36 publications

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“…50 mM Na ϩ or K ϩ showed no inhibition. The monovalent cations Cs ϩ and Tl ϩ have been shown to bind to the ammonium binding site of glutamine synthetase (22). A concentration of 50 mM Cs ϩ was required for full inhibition of AmtB activity (data not shown), but Tl ϩ effected complete inhibition at 0.5 mM (Fig.…”

Section: Resultsmentioning

confidence: 91%

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

Javelle

Lupo

Ripoche

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

161

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The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic ''gate'' formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH4 ؉ deprotonation takes place at S1 before NH3 conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

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Section: Resultsmentioning

confidence: 91%

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

Javelle

Lupo

Ripoche

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

161

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“…The increase in the number of activated astrocytes would be expected to increase the uptake of glutamate. In astrocytes, glutamine synthetase catalyzes conversion of glutamate to glutamine (40). Because there is no detectable ERβ in astrocytes, the change in the number of reactive astrocytes seen upon LY3201 treatment is likely not caused by direct action of the ligand on astrocytes.…”

Section: Discussionmentioning

confidence: 99%

Reduction of dendritic spines and elevation of GABAergic signaling in the brains of mice treated with an estrogen receptor β ligand

Tan

Dai

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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An estrogen receptor (ER) β ligand (LY3201) with a preference for ERβ over ERα was administered in s.c. pellets releasing 0.04 mg/d. The brains of these mice were examined 3 d after treatment had begun. Although estradiol-17β is known to increase spine density and glutaminergic signaling, as measured by Golgi staining, a clear reduction in spines was evident on the dendritic branches in LY3201-treated mice but no morphological alteration and no difference in the number of dendritic spines on dendritic stems were observed. In the LY3201-treatment group, there was higher expression of glutamic acid decarboxylase (GAD) in layer V of cortex and in the CA1 of hippocampus, more GAD + terminals surrounding the pyramidal neurons and less glutamate receptor (NMDAR) on the neurons in layer V. There were no alterations in expression of Iba1 or in Olig2 or CNPase. However, GFAP + astrocytes were increased in the LY3201-treatment group. There were also more projections characteristic of activated astrocytes and increased expression of glutamine synthetase (GS). No expression of ERβ was detectable in the nuclei of astrocytes. Clearly, LY3201 caused a shift in the balance between excitatory and inhibitory neurotransmission in favor of inhibition. This shift was due in part to increased synthesis of GABA and increased removal of glutamate from the synaptic cleft by astrocytes. The data reveal that treatment with a selective ERβ agonist results in changes opposite to those reported in estradiol-17β-treated mice and suggests that ERα and ERβ play opposing roles in the brain.neurotransmitter | glutamate toxicity | cerebral cortex | hippocampus O f the two estrogen receptors (ERs), ERα appears to be the major player in regulating the reproductive system, whereas both receptors ERα and ERβ are involved in regulation of mood and affective disorders (1, 2). Several studies show that ERα activation is anxiogenic (3-5), whereas ERβ activation is anxiolytic and controls expression of fear and anxiety-like behaviors (6-9). Not surprisingly, estradiol (E2), which binds equally well to ERα and ERβ, has unpredictable effects on abnormal psychiatric behaviors, depending on dose, treatment regimens, or animal models (10-13). In hippocampal cultures both exogenous (E2) treatment and locally synthesized E2 up-regulate ERα and down-regulate ERβ expression (14). Thus, E2 has unpredictable effects on the balance between anxiogenic and anxiolytic effects, in part because E2 has opposite effects on ERα and ERβ (15). Using subtype-selective ligands, we can begin to study the outcome of selective activation or inhibition of a particular receptor subtype involved in different behaviors.Spines and the synapses located on neurons are the sites at which neuronal communication occurs and are essential for complex behavioral phenomena and cognition. In the mature CNS, alterations in synapse structure and function continue to be a dynamic process that is of fundamental importance to learning and memory as well as other adaptive abilities of the brain. Studies...

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“…Ammonia that bypasses this primary fate is subsequently 'picked up' and detoxified by glutamine synthetase (GS), an enzyme found in the hepatocytes surrounding the hepatic vein (as well as in muscle and astroglial cells), which catalyses the conversion of ammonia and glutamate to glutamine. 23 Whilst the liver is critical in the homeostatic control of blood ammonia levels, other organs such as the brain, muscle and kidney are also known to play a role in regulating them. Insult to the liver, whether acute or chronic in nature, reduces its capacity to metabolize ammonia and this exerts an ammonia burden on extrahepatic tissues which can result in hyperammonaemia up to five times that of normal blood ammonia levels.…”

Section: Interorgan Ammonia Metabolism and Regulationmentioning

confidence: 99%

Pathogenesis of Hepatic Encephalopathy: Role of Ammonia and Systemic Inflammation

Aldridge

Tranah

Shawcross

2015

Journal of Clinical and Experimental Hepatology

235

190

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The syndrome we refer to as Hepatic Encephalopathy (HE) was first characterized by a team of Nobel Prize winning physiologists led by Pavlov and Nencki at the Imperial Institute of Experimental Medicine in Russia in the 1890's. This focused upon the key observation that performing a portocaval shunt, which bypassed nitrogen-rich blood away from the liver, induced elevated blood and brain ammonia concentrations in association with profound neurobehavioral changes. There exists however a spectrum of metabolic encephalopathies attributable to a variety (or even absence) of liver hepatocellular dysfunctions and it is this spectrum rather than a single disease entity that has come to be defined as HE. Differences in the underlying pathophysiology, treatment responses and outcomes can therefore be highly variable between acute and chronic HE. The term also fails to articulate quite how systemic the syndrome of HE can be and how it can be influenced by the gastrointestinal, renal, nervous, or immune systems without any change in background liver function. The pathogenesis of HE therefore encapsulates a complex network of interdependent organ systems which as yet remain poorly characterized. There is nonetheless a growing recognition that there is a complex but influential synergistic relationship between ammonia, inflammation (sterile and non-sterile) and oxidative stress in the pathogenesis HE which develops in an environment of functional immunoparesis in patients with liver dysfunction. Therapeutic strategies are thus moving further away from the traditional specialty of hepatology and more towards novel immune and inflammatory targets which will be discussed in this review. ( J CLIN EXP HEPATOL 2015;5:S7-S20) H epatic encephalopathy (HE) is the term used to encapsulate the broad spectrum of neuropsychiatric disturbances associated with both acute and chronic liver failure (ALF and CLF, respectively), as well as porto-systemic bypass in the absence of hepatocellular disease. The clinical manifestations of HE can be extremely heterogeneous in nature, with symptoms presenting anywhere on a continuum spanning from seemingly normal cognitive performance, right the way through to states of confusion, stupor and coma. In between these extremes, patients with HE may exhibit signs such as inattentiveness, blunted affect, impairment of memory or reversal of the sleep-wake cycle, as well as physical manifestations such as tremor, myoclonus, asterixis and deep tendon hyperreflexia.ALF is defined by the onset of coagulopathy alongside any degree of encephalopathy in patients with no evidence of pre-existing liver disease. 1 The presence of HE in those with ALF is prognostic, with up to a quarter of cases developing raised intracranial pressure. 2 Patients presenting with ALF are at risk of developing its cardinal, lifethreatening feature, cerebral edema. Left untreated, cerebral edema can rapidly progress to cause herniation of the uncus through the falx cerebri, leading to compression of the brainstem and, ultimately, death. H...

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Discovery of the ammonium substrate site on glutamine synthetase, A third cation binding site

Cited by 94 publications

References 36 publications

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

Reduction of dendritic spines and elevation of GABAergic signaling in the brains of mice treated with an estrogen receptor β ligand

Pathogenesis of Hepatic Encephalopathy: Role of Ammonia and Systemic Inflammation

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