Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca<sup>2+</sup> Chelators

Tymianski, Michael; Spigelman, Igor; Zhang, Liang; Carlen, Peter L.; Tator, Charles H.; Charlton, Milton P.; Wallace, M. Christopher

doi:10.1038/jcbfm.1994.122

Cited by 72 publications

(44 citation statements)

References 87 publications

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“…To directly test the role of endosomal calcium in RuV fusion, we determined the ability of BAPTA-AM to block virus infection. BAPTA-AM diffuses freely across membranes but requires activation to BAPTA by intracellular esterases and thus chelates only intracellular calcium (39)(40)(41). BAPTA-AM has been found to strongly chelate intracellular calcium stores within 2 min of cell treatment (47).…”

Section: Discussionmentioning

confidence: 99%

“…However, these results could not differentiate between effects on E1 membrane insertion at the cell surface and those within the endosome compartment. To address the location of calcium-dependent insertion, we took advantage of the membrane-permeant calcium chelator BAPTA-AM (39)(40)(41). This chelator is inactive until its acetoxymethyl group is removed by intracellular esterases, thus trapping charged active BAPTA within the cell, where it specifically chelates intracellular calcium.…”

Section: The Configuration Of Camentioning

confidence: 99%

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Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Dubé

Etienne

Fels

et al. 2016

J Virol

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The E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH. IMPORTANCE Rubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein.Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment. R ubella virus (RuV) is a small enveloped single-stranded RNA virus and the sole member of the Rubivirus genus. Rubivirus and alphaviruses together comprise the Togaviridae (for an overview, see reference 1). While alphaviruses are generally transmitted by mosquito vectors, RuV spreads by airborne transmission between humans, the only known host (2). RuV causes a mild childhood disease commonly referred to as German measles (for a review, see references 1 and 3). However, RuV is able to cross the placental barrier, and infection of pregnant women, particularly during the first trimester, can cause miscarriage, stillbirth, or severe fetal malformations known as congenital rubella syndrome (CRS) (1, 4). While vaccination has essentially eliminated RuV disease and CRS in the Ame...

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Section: Discussionmentioning

confidence: 99%

Section: The Configuration Of Camentioning

confidence: 99%

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Dubé

Etienne

Fels

et al. 2016

J Virol

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“…We hypothesized that transient [Ca 2ϩ ] i mobilization by the non-genomic activity of testosterone affects Duox1-dependent H 2 O 2 generation. BAPTA-AM is known to scavenge intracellular Ca 2ϩ (26). The pretreatment of keratinocytes with BAPTA-AM as an intracellular Ca 2ϩ scavenger resulted in significantly reduced testosterone-induced H 2 O 2 generation, compared with control cells (Fig.…”

Section: Connection Between the Non-genomic Activity Of Testosterone mentioning

confidence: 99%

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Choi

Kim

et al. 2014

Journal of Biological Chemistry

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Background:The molecular mechanisms underlying the non-genomic activities of testosterone in keratinocytes are unknown. Results: Testosterone stimulates Duox1 activity through GPRC6A leading to cell death in skin keratinocyte. Conclusion: These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis through Duox1-induced H 2 O 2 generation. Significance: These results provide a novel signaling cascade of testosterone-mediated redox regulation in keratinocytes.

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“…The chelators may act postsynaptically by attenuating neurotoxic NMDA-mediated cytosolic calcium loading (Tymianski et al, 1994a) and /or presynaptically by attenuating excitatory neurotransmitter release (Tymianski et al, 1994b;Ouanounou et al, 1996;Spigelman et al, 1996). Thus, we first examined the role of NMDA receptor activation in OGD toxicity in the present preparation.…”

Section: Ogd Neurotoxicity Is Mediated Partially By Nmda Receptor Actmentioning

confidence: 99%

“…If synaptic overactivity mediates anoxic cell death (Kass and Lipton, 1982;Rothman, 1983Rothman, , 1984, then protection by Ca 2ϩ buffers, which attenuate synaptic transmitter release in many preparations (Adler et al, 1991;Niesen et al, 1991;Tymianski et al, 1994b;Ouanounou et al, 1996;Spigelman et al, 1996), may occur by this mechanism. However, exogenous Ca 2ϩ buffers also attenuate postsynaptic cytosolic Ca 2ϩ increases in neurons (Tymianski et al, 1993c(Tymianski et al, , 1994a.…”

Section: ؉ Buffers Are Ineffective As Neuroprotectants When Transmitmentioning

confidence: 99%

Mechanisms and Effects of Intracellular Calcium Buffering on Neuronal Survival in Organotypic Hippocampal Cultures Exposed to Anoxia/Aglycemia or to Excitotoxins

Abdel-Hamid¹,

Tymianski

1997

J. Neurosci.

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117

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Neuronal calcium loading attributable to hypoxic/ischemic injury is believed to trigger neurotoxicity. We examined in organotypic hippocampal slice cultures whether artificially and reversibly enhancing the Ca 2ϩ buffering capacity of neurons reduces the neurotoxic sequelae of oxygen-glucose deprivation (OGD), whether such manipulation has neurotoxic potential, and whether the mechanism underlying these effects is preor postsynaptic. Neurodegeneration caused over 24 hr by 60 min of OGD was triggered largely by NMDA receptor activation and was attenuated temporarily by pretreating the slices with cell-permeant Ca 2ϩ buffers such as 1,2 bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetra-acetic acid acetoxymethyl ester (BAPTA-AM). This pretreatment produced a transient, reversible increase in intracellular buffer content as demonstrated autoradiographically using slices loaded with 14 C-BAPTA-AM and by confocal imaging of slices loaded with the BAPTA-AM analog calcium green-acetoxymethyl ester (AM). The time courses of 14 C-BAPTA retention and of neuronal survival after OGD were identical, indicating that increased buffer content is necessary for the observed protective effect. Protection by Ca 2ϩ buffering originated presynaptically because BAPTA-AM was ineffective when endogenous transmitter release was bypassed by directly applying NMDA to the cultures, and because pretreatment with the low Ca 2ϩ affinity buffer 2-aminophenol-N,N,O-triacetic acid acetoxymethyl ester, which attenuates excitatory transmitter release, attenuated neurodegeneration. Thus, in cultured hippocampal slices, enhancing neuronal Ca 2ϩ buffering unequivocally attenuates or delays the onset of anoxic neurodegeneration, likely by attenuating the synaptic release of endogenous excitatory neurotransmitters (excitotoxicity).

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Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca²⁺ Chelators

Cited by 72 publications

References 87 publications

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Mechanisms and Effects of Intracellular Calcium Buffering on Neuronal Survival in Organotypic Hippocampal Cultures Exposed to Anoxia/Aglycemia or to Excitotoxins

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Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca2+ Chelators

Cited by 72 publications

References 87 publications

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Calcium-Dependent Rubella Virus Fusion Occurs in Early Endosomes

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Mechanisms and Effects of Intracellular Calcium Buffering on Neuronal Survival in Organotypic Hippocampal Cultures Exposed to Anoxia/Aglycemia or to Excitotoxins

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Mechanism of Action and Persistence of Neuroprotection by Cell-Permeant Ca²⁺ Chelators