The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function

Marshall, John; Molloy, Raymond; Moss, Guy W. J.; Howe, James R.; Hughes, Thomas E.

doi:10.1016/0896-6273(95)90279-1

Cited by 239 publications

(138 citation statements)

References 12 publications

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“…A similar NR1 C-terminal GFP fusion has been found to form functional channels when coexpressed with NR2 subunits in heterologous cells (Marshall et al, 1995), but its localization has not yet been reported in neurons. Hippocampal neurons were transfected at plating and imaged at 15-16 d (Fig.…”

Section: Synaptic Targeting Of Nmda Receptors Can Be Visualized In LImentioning

confidence: 99%

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

2001

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Chronic activity blockade increases synaptic levels of NMDA receptor immunoreactivity in hippocampal neurons. We show here that blockade-induced synaptic NMDA receptors are functional and mediate enhanced excitotoxicity in response to synaptically released glutamate. Activity blockade increased the cell surface association of NMDA receptors. Blockade-induced synaptic targeting of NMDA receptors did not require protein synthesis but required phosphorylation and specifically cAMPdependent protein kinase (PKA). Furthermore, activation of PKA was sufficient to induce synaptic targeting of NMDA receptors regardless of receptor activity status. These results implicate PKA activity downstream of receptor blockade as a mediator of enhanced synaptic transport or stabilization of NMDA receptors. Synaptic clustering of NR1-green fluorescent protein was observed in living neurons in response to NMDA receptor and cAMP phosphodiesterase antagonists and occurred gradually over the course of a day. This pathway represents a cellular mechanism for synaptic homeostasis and is likely to function in metaplasticity, long-term regulation of the ability of a synapse to undergo potentiation or depression. Key words: NMDA receptor; synaptogenesis; activity; synaptic clustering; excitotoxicity; subcellular localization; hippocampus; NR1-GFPThe NMDA-type glutamate receptor plays a central role in circuit development, memory formation, and many forms of synaptic plasticity in the mammalian brain. The NMDA receptor is composed of the essential NR1 subunit and one or more of the modulatory NR2A-D and NR3 subunits (Nakanishi, 1992;Seeburg, 1993;Mori and Mishina, 1995). NMDA receptor channel opening requires ligand binding (by presynaptic glutamate release) and removal of Mg 2ϩ block (by postsynaptic depolarization), thus conferring on the NMDA receptor the ability to function as a molecular coincidence detector (Mayer et al., 1984). Through its Ca 2ϩ permeability, NMDA receptor function is linked with many downstream signal transducing pathways in the neuron. The magnitude and kinetics of calcium elevation at the synapse are thought to be major determinants of long-term effects on synaptic efficacy (Lisman, 1989;Abraham and Bear, 1996).The level of NMDA receptor function at the synapse critically regulates brain function and cell survival. Mice expressing 5% of normal levels of NR1 exhibit increased motor activity, stereotypy, and deficits in social and sexual interactions, behaviors associated with schizophrenia (Mohn et al., 1999). Deletion of NR1 targeted postnatally selectively to CA1 of the hippocampus results in mice that are viable but deficient in spatial learning and formation of temporal memory (Tsien et al., 1996;Huerta et al., 2000). In contrast, overactivation of NMDA receptors contributes substantially to neuronal death during epilepsy, stroke, trauma, and neurodegenerative disorders (McDonald and Johnston, 1990;Choi, 1994;Rothman and Olney, 1995;During et al., 2000).NMDA receptor function is regulated during development and by ...

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Section: Synaptic Targeting Of Nmda Receptors Can Be Visualized In LImentioning

confidence: 99%

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

2001

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“…Note the partial recovery and the similar run-down after 24 h. chimeras has been observed with other receptor GFP constructs (1,26), some of our findings were unexpected, and are in marked contrast to observations on other ligand-gated receptors fused to GFP. For example, such fusion does not seem to alter greatly the properties of N-methyl-D-aspartate (NMDA), glycine, GABA A , and GABA1 receptors (1,(26)(27)(28). Instead, in this work we show that the kinetics of AcCho-current inactivation change appreciably after receptor fusion with GFP, in such a way that desensitization of the chimeric wt␣7-GFP receptor becomes slower, whereas that of the chimeric mut␣7-GFP receptor becomes faster than their respective non-GFP controls.…”

Section: Functional Expression Of Human Wt␣7-gfp Receptorsmentioning

confidence: 99%

Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

Palma

Mileo²,

Martı́nez-Torres

et al. 2002

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

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The functional properties and cellular localization of the human neuronal ␣7 nicotinic acetylcholine (AcCho) receptor (␣7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mut␣7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wt␣7 receptors decay much faster than those elicited by the mut␣7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated ␣7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable ''run-down'' of the AcCho currents generated by mut␣7-GFP receptors, whereas those of the wt␣7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mut␣7-GFP oocytes was accompanied by a marked decrease of ␣-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wt␣7 and mut␣7 receptors provides powerful tools to study the distribution and function of ␣7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. F luorescent tagging of cellular proteins is a very useful, and aesthetically pleasant, method for studying the intracellular traffic and distribution of many proteins, including neurotransmitter receptors. It is commonly assumed that the tagged proteins function in exactly the same way as the native nontagged proteins. To determine whether that is the case for neurotransmitter receptors, we chose to study two human receptors that are homomeric: the GABA1 (1) and now the ␣7 nicotinic receptors.The neuronal ␣7 nicotinic acetylcholine receptor (␣7-AcChoR) is widely expressed in the central nervous system and, being located mainly at nerve terminals, it is believed to be involved in regulating the neurotransmitter release that mediates fast cholinergic neurotransmission (2, 3). In heterologous expression systems, including the Xenopus oocyte, the ␣7-subunit forms functional homomeric AcChoRs that show an unusually fast desensitization and high permeability to Ca 2ϩ (4-8). Even though it is not yet clear to what extent the ␣7 homomeric composition also applies in vivo, we and others have found it of interest to investigate the functional profile of homomeric ␣7 receptors, because they may be involved in the pathogenesis of many neurological disorders (9-16).For this study we constructed a chimera in which the enhanced form of the green fluorescent protein (GFP) was fused to the C...

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“…The expression of this intrinsically fluorescent protein in mammalian cells has generated a considerable amount of interest in the use of this molecule to 'tag' intracellular proteins which can be monitored in situ [14][15][16][17]. There are some reports of the successful expression of GFPfusion proteins in mammalian cells, for example with the NMDA receptor [18], MAP4 [19] and cyclins [20]. However, the extent to which GFP, which is a relatively large (27 kDa) protein, might interfere with the normal regulatable trafficking of a protein in mammalian cells has not been adequately addressed.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein

et al. 1996

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Insulin increases glucose uptake by promoting the translocation of the GLUT4 isoform of glucose transporters to the plasma membrane. We have studied this process in living single cells by fusing green fluorescent protein (GFP) to the Nterminus (GFP.GLUT4) or C-terminus (GLUT4.GFP) of GLUT4. Both chimeras were expressed in a perinuclear compartment of CHO cells, and in a vesicular distribution through the cytosol. Insulin promoted an increase in plasma membrane fluorescence as a result of net translocation of the chimeras to the cell surface. GLUT4-GFP, but not GFP.GLUT4, was re-internalised upon the removal of insulin suggesting that a critical internalisation signal sequence exists in the N-terminus of GLUT4. The use of GFP thus allows an analysis of GLUT4 trafficking in single living cells.

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The jellyfish green fluorescent protein: A new tool for studying ion channel expression and function

Cited by 239 publications

References 12 publications

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein

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