Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale.

Wieboldt, Raymond; Gee, Kyle R.; Niu, Li; Ramesh, Dharavath; Carpenter, Barry K.; Hess, George P.

doi:10.1073/pnas.91.19.8752

Cited by 184 publications

(233 citation statements)

References 30 publications

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“…We report here the investigation of rapid transporter dynamics on a submillisecond time scale by applying the technique of laser-pulse photolysis of caged glutamate (7)(8)(9). The results are consistent with a sequential shuttle model, in which the electrogenic Na ϩ -dependent glutamate translocation occurs within milliseconds and is preceded by submillisecond binding of glutamate to the transporter.…”

supporting

confidence: 62%

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds

Grewer

Watzke

Wiessner

et al. 2000

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

172

323

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The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laserpulse photolysis technique of caged glutamate with a time resolution of 100 s. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substratecoupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system.

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supporting

confidence: 62%

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds

Grewer

Watzke

Wiessner

et al. 2000

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

172

323

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“…In this paradigm, the animals can be trained and behavior monitored while the head capsule is open and the brain is exposed. This condition makes the brain accessible for locally and temporally defined release of glutamate by photolytic stimulation of caged glutamate (Wieboldt et al, 1994). We demonstrate that photorelease of glutamate in the mushroom bodies (MBs) facilitates olfactory memory.…”

Section: Introductionmentioning

confidence: 99%

Focal and Temporal Release of Glutamate in the Mushroom Bodies Improves Olfactory Memory inApis mellifera

Locatelli

Bundrock²,

Müller³

2005

J. Neurosci.

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In contrast to vertebrates, the role of the neurotransmitter glutamate in learning and memory in insects has hardly been investigated. The reason is that a pharmacological characterization of insect glutamate receptors is still missing; furthermore, it is difficult to locally restrict pharmacological interventions. In this study, we overcome these problems by using locally and temporally defined photo-uncaging of glutamate to study its role in olfactory learning and memory formation in the honeybee, Apis mellifera. Uncaging glutamate in the mushroom bodies immediately after a weak training protocol induced a higher memory rate 2 d after training, mimicking the effect of a strong training protocol. Glutamate release before training does not facilitate memory formation, suggesting that glutamate mediates processes triggered by training and required for memory formation. Uncaging glutamate in the antennal lobes shows no effect on memory formation. These results provide the first direct evidence for a temporally and locally restricted function of glutamate in memory formation in honeybees and insects.

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“…It was thought that since the appearance of ATP from NPE-caged ATP was concomitant with the decay of its aci-nitro intermediate 14 , that the decay of this species could be used as an indicator of the release of other caged compounds (for example, IP 3 (ref. 12 ) and glutamate 9 ). Rigorous study of the kinetics of model ortho-nitrobenzyl (ortho-NB)|[ndash]|caged compounds (caged ethers) has however shown this is not always the case 66 .…”

Section: Chemical Considerationsmentioning

confidence: 99%

Caged compounds: photorelease technology for control of cellular chemistry and physiology

2007

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Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group (Fig. 1). Illumination results in a concentration jump of the biologically active molecule that can bind to its cellular receptor, switching on (or off) the targeted process. Virtually every kind of signaling molecule or second messenger, of every size| [mdash]|from protons to proteins| [mdash]|has been caged 1 .Why are caged compounds so useful? A single component of cellular chemistry can control the function of a cell, and such cellular regulation can be temporally or spatially defined, intracellular or extracellular, and amplitude-or frequency-modulated 2 . Photomanipulation of cellular chemistry using caged compounds provides a uniquely powerful means to interact with such cellular dynamics, as it can touch upon any one of the above dimensions. Thus, since light passes through cell membranes, uncaging can rapidly release a biomolecule in an intracellular compartment. This space is not readily accessible to many second messengers (for example, inositol-1,4,5-trisphosphate (IP 3 ), ATP, Ca 2+ , cAMP, cGMP) when they are applied to cells externally, as their charge makes them impermeable to the plasma membrane. Furthermore, uniform illumination results in release throughout the cytosol, or the release can be localized by focusing the uncaging beam on one part of a cell. Likewise, extracellular uncaging of neurotransmitters and hormones is tunable, allowing stimulation of many neurons simultaneously or of single synapses| [mdash]|by global or focused illumination, respectively. Light cannot only be directed, but also modulated in time andCorrespondence should be addressed to Graham C R Ellis-Davies (cagedca@hotmail.com). (Fig. 2) has been widely used to study many Ca 2+ -controlled processes. In particular, secretory processes in neuronal and non-neuronal cells have been extensively studied with caged calcium 22 . Rapid uncaging of glutamate using two-photon photolysis is particularly useful for the rational stimulation of visually identified synapses in complex tissue preparations such as acutely isolated brain slices from the hippocampus 23 (Fig. 2b). Finally, uncaging of mRNA in vivo in zebrafish is an excellen...

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Photolabile precursors of glutamate: synthesis, photochemical properties, and activation of glutamate receptors on a microsecond time scale.

Cited by 184 publications

References 30 publications

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds

Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds

Focal and Temporal Release of Glutamate in the Mushroom Bodies Improves Olfactory Memory inApis mellifera

Caged compounds: photorelease technology for control of cellular chemistry and physiology

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