How Fast Does the γ-Aminobutyric Acid Receptor Channel Open? Kinetic Investigations in the Microsecond Time Region Using a Laser-Pulse Photolysis Technique

Jayaraman, Vasanthi; Thiran, Shalita; Hess, George P.

doi:10.1021/bi990454c

Cited by 28 publications

(38 citation statements)

References 38 publications

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“…Since the first demonstration of its use in the kinetic investigation of the channel-opening mechanism for the muscle-type nicotinic acetylcholine receptor (38), the laser-pulse photolysis technique has been applied to several other receptor types (31,32,65), including the GluR6Q kainate receptor (55) and the GluR2Q flip AMPA receptor (56). It has further enabled kinetic investigations of the mechanism of drug-receptor interactions in the microsecond-to-millisecond time domain where the receptors were in their functional forms before receptor desensitization (66 -69).…”

Section: Discussionmentioning

confidence: 99%

How Fast Does the GluR1Qflip Channel Open?

Niu

2004

Journal of Biological Chemistry

152

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Opening of a ligand-gated ion channel is the step at which the binding of a neurotransmitter is transduced into the electrical signal by allowing ions to flow through the transmembrane channel, thereby altering the postsynaptic membrane potential. We report the kinetics for the opening of the GluR1Q flip channel, an ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit of the ionotropic glutamate receptors. Using a laser-pulse photolysis technique that permits glutamate to be liberated photolytically from ␥-O-(␣-carboxy-2-nitrobenzyl)glutamate (caged glutamate) with a time constant of ϳ30 s, we show that, after the binding of glutamate, the channel opened with a rate constant of (2.9 ؎ 0.2) ؋ 10 4 s ؊1 and closed with a rate constant of The rate at which a ligand-gated ion channel opens is important to know because it has major implications in signal transmission and regulation. First, knowing the constants for the channel-opening rate will allow one to predict more quantitatively the time course of the open channel form of the receptor as a function of neurotransmitter or ligand concentration, which determines the transmembrane voltage change and in turn controls synaptic neurotransmission. Second, that knowledge will provide clues for mechanism-based design of compounds to regulate receptor function more effectively. Third, characterizing the effect of structural variations on the rate constants for channel opening will offer a test of the function, which is relevant to the time scale on which the receptor is in the open channel form, rather than in the desensitized form, i.e. ligand-bound, but closed channel form. Examples of structural variations include those due to RNA editing, RNA splicing, and site-specific mutations for investigating the structurefunction relationship. Finally, knowing the channel-opening rate constants will be required to understand quantitatively the integration of nerve impulses that arrive at a chemical synapse or that originate from the same synapse, but from different receptors responding to the same chemical signals (neurotransmitters) such as glutamate.We report here the kinetics for the opening of the GluR1Q flip receptor channel. GluR1 is one of the four subunits of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

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Section: Discussionmentioning

confidence: 99%

How Fast Does the GluR1Qflip Channel Open?

Niu

2004

Journal of Biological Chemistry

152

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“…The question we asked was: Can additional information about the receptor-mediated reactions be obtained by using presteadystate kinetic techniques? Recently, presteady-state kinetic techniques that are suitable for measuring receptor-mediated reactions on cell surfaces in the millisecond-to-microsecond time region were developed (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The time resolution of the laser-pulse photolysis technique (23)(24)(25)(26)) is sufficient to investigate the reaction before the channel has opened.…”

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confidence: 99%

Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801

Hess

Ulrich

Breitinger

et al. 2000

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

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Nicotinic acetylcholine receptors (AChR) belong to a family of proteins that form ligand-gated transmembrane ion channels. They are involved in the fast transmission of signals between cells and the control of intercellular communication in the nervous system. A variety of therapeutic agents and abused drugs, including cocaine, inhibit the AChR and monoamine transporters and interfere with nervous system function. Here we describe a mechanism-based approach to prevent this inhibition. We had previously developed presteady-state kinetic (transient kinetic) techniques, with microsecond-to-millisecond time resolutions, for investigations of reactions on cell surfaces that allow one to determine the effects of inhibitors not only on the channel-opening probability but also on the opening and closing rates of the AChR channel. The transient kinetic measurements led to two predictions. T he nicotinic acetylcholine receptor (AChR) is the prototypical member of a family of structurally related membrane proteins, the ligand-gated ion channels (1). These proteins regulate intercellular communication between the approximately 10 12 cells of the mammalian nervous system, a process considered essential for brain function (2). Many therapeutic agents and abused drugs affect their function (3). For instance, the AChR is inhibited by the anticonvulsant MK-801 [(ϩ)Ϫdizocilpine] (4-6) and by several abused drugs, including cocaine (7-9). Cocaine affects more than three million people annually in the United States alone, at an estimated cost to society of more than 100 billion dollars.Understanding the mechanism of the AChR and its inhibition is a longstanding and challenging problem (10) with major implications for medicine and drug addiction (11-12). Two decades ago, single-channel current-recording (13) measurements led to the proposal of a simple and generally accepted mechanism in which inhibitors enter the open channel and block it (14-17) (the channel-blocking mechanism, Mechanism A in Fig. 1). Although several variations of this open-channelblocking mechanism have been proposed, including the conversion of an inhibitor-bound closed-channel conformation to a blocked open-channel form (18-21) (Mechanism B, Fig. 1), the open-channel-blocking mechanism, based mainly on singlechannel current or other steady-state kinetic measurements (14-21), has met the test of time during the last 20 years. In the techniques used for those measurements, the channel-activating ligand is in quasi equilibrium (steady state) with the receptor. The question we asked was: Can additional information about the receptor-mediated reactions be obtained by using presteadystate kinetic techniques? Recently, presteady-state kinetic techniques that are suitable for measuring receptor-mediated reactions on cell surfaces in the millisecond-to-microsecond time region were developed (22-31). The time resolution of the laser-pulse photolysis technique (23-26) is sufficient to investigate the reaction before the channel has opened. One can, therefore, obtain info...

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Section: Discussionmentioning

confidence: 99%

“…7, 200746, No. 7, 2033 brain, as it determines such basic properties as temporal precision and reliability (64,65) Table 3). The comparison of the channel opening kinetics among these receptor subunits reveals clear differences in the channel opening and channel closing rate constants.…”

Section: Glur3flipmentioning

confidence: 99%

“…a faster channel-closing rate) of a kainate or an AMPA channel compared with the muscle nicotinic acetylcholine receptor is often observed. Moreover, the brief opening duration of such a channel makes it even more difficult to analyze the "flickering" in open channel bursts to investigate the mechanism of inhibition (63,64).Since the first demonstration of its use in the kinetic investigation of the channel-opening mechanism for the muscle-type nicotinic acetylcholine receptor (38), the laser-pulse photolysis technique has been applied to several other receptor types (31, 32,65), including the GluR6Q kainate receptor (55) and the GluR2Q flip AMPA receptor (56). It has further enabled kinetic investigations of the mechanism of drug-receptor interactions in the microsecond-to-millisecond time domain where the receptors were in their functional forms before receptor desensitization (66 -69).…”

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Glutamate Receptor Aptamers and ALS

Niu¹

2008

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. REPORT DATE 07-01-2008 REPORT TYPE PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBERUniversity at Albany, SUNY Albany, New York 12222 SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTES ABSTRACTExcitotoxicity is one of the leading causes for amyotrophic lateral sclerosis (ALS). Our goal was to develop a novel class of powerful aptamer-based, anti-excitotoxic inhibitors against GluR2Qflip, a key AMPA receptor subunit that controls the calcium permeability and mediates excitotoxicity. An aptamer is a single-stranded nucleic acid that directly inhibits a protein's function by folding into a specific tertiary structure that dictates high-affinity binding to the target protein.To date, we have identified two classes of aptamers (i.e. competitive and noncompetitive aptamers) against GluR2Qflip, by using a molecular biology approach called systematic evolution of ligands by exponential enrichment (SELEX). These aptamers are water soluble and have a nanomolar affinity against GluR2Qflip. Their inhibitory properties rival those of any existing small, chemical inhibitors. We are continuing to work with these aptamers towards developing them into anti-excitotoxic drugs for treating patients with ALS, including those Gulf War veterans suffering from ALS. SUBJECT TERMS INTRODUCTIONStudies of Gulf War veterans with ALS have suggested a link of Gulf War service to this fatal neurodegenerative disease. Although the pathogenesis of the war-related ALS is unknown, the excitotoxicity or excessive excitatory neurotransmission, mainly mediated by the AMPA-type (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) glutamate receptors, may play a central role in the selective motor neuron death in ALS. Thus, developing AMPA receptor inhibitors to control the receptor-mediated neurodegeneration has been a long-pursued therapeutic effort. To d...

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How Fast Does the γ-Aminobutyric Acid Receptor Channel Open? Kinetic Investigations in the Microsecond Time Region Using a Laser-Pulse Photolysis Technique

Cited by 28 publications

References 38 publications

How Fast Does the GluR1Qflip Channel Open?

How Fast Does the GluR1Qflip Channel Open?

Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801

Glutamate Receptor Aptamers and ALS

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