PROTEIN CONFORMATIONAL CHANGES IN THE μs TIME REGION INVESTIGATED WITH A LASER PULSE PHOTOLYSIS TECHNIQUE11We gratefully acknowledge financial support by grants (GM04942 and RR04804) awarded to G.P.H. by the National Institutes of Health.

Billington, Andrew P.; Matsubara, Naoko; Webb, Watt W.; Hess, George P.

doi:10.1016/b978-0-12-058756-8.50047-x

Cited by 3 publications

(2 citation statements)

References 27 publications

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“…This had two purposes: (1) to monitor recording stability for the LaPP study and (2) to determine if the RCNB-caged glutamate was contaminated with free glutamic acid. The physical arrangement of the cell, U-tube, and fiber optic for the LaPP experiments has been described in detail (13,31,32). Before and after each LaPP experiment, the energy of the laser was determined by using a Gentec joule meter.…”

Section: Chemicalsmentioning

confidence: 99%

Mechanism of Glutamate Receptor-Channel Function in Rat Hippocampal Neurons Investigated Using the Laser-Pulse Photolysis (LaPP) Technique

Nowak

Gee

et al. 2002

Biochemistry

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Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).

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Section: Chemicalsmentioning

confidence: 99%

Mechanism of Glutamate Receptor-Channel Function in Rat Hippocampal Neurons Investigated Using the Laser-Pulse Photolysis (LaPP) Technique

Nowak

Gee

et al. 2002

Biochemistry

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“…The observed current was corrected for receptor desensitization that occurs during equilibration of the cell surface receptors with the ligands in the flowing solution (25,26). The laser-pulse photolysis experiments were performed as described by Billington et al (27). The photocleavage of the caged carbamoylcholine was initiated with a pulse of laser light generated by a Lumonics nitrogen excimer laser (λ ) 337 nm).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Nicotinic Acetylcholine Receptor by Philanthotoxin-343: Kinetic Investigations in the Microsecond Time Region Using a Laser-Pulse Photolysis Technique

1999

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The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.

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On the Mechanism of Inhibition of the Nicotinic Acetylcholine Receptor by the Anticonvulsant MK-801 Investigated by Laser-Pulse Photolysis in the Microsecond-to-Millisecond Time Region

Grewer

Hess

1999

Biochemistry

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The mechanism of inhibition of the muscle nicotinic acetylcholine receptor is of interest because of the many drugs which are known to modify its function. The laser-pulse photolysis technique, using a photolabile, biologically inert ligand (caged carbamoylcholine) for the nicotinic acetylcholine receptor, and BC3H1 cells have been used to investigate the mechanism of inhibition of the receptor by MK-801 [(+)-dizocilpine] in the microsecond-to-millisecond time region. MK-801 is an anticonvulsant and a known inhibitor of the N-methyl-D-aspartate and nicotinic acetylcholine receptors. Both the chemical kinetic and the single-channel current-recording measurements reported here indicate the existence of two inhibition processes, one occurring within 50 ms and the other within about 1 s of equilibration of the receptor with the inhibitor. Unless stated otherwise, here we characterize the receptor inhibition observed when MK-801 is equilibrated with the receptor for only 50 ms. We determined the effect of MK-801 on the concentration of the open receptor-channels and the apparent dissociation constant of the inhibitor from the closed-channel (KI(obs) = 180 microM) and open-channel ( = 950 microM) forms. Within a few milliseconds after inhibitor binding, decreases to about 100 microM, due to an inhibitor-induced isomerization to an inactive receptor form. A mechanism that incorporates the new results is proposed. It includes the formation of an ion-conducting receptor:inhibitor complex with a channel-opening equilibrium constant that is unfavorable compared to the open-channel receptor form in the absence of inhibitor. In the MK-801 concentration range of 0-500 microM, this mechanism accounts for the observed MK-801-induced decrease in the concentration of open channels. At high concentrations of carbamoylcholine, when the receptor is mainly in the open-channel form, the conducting receptor:inhibitor complex isomerizes to a nonconducting state with a rate constant of about 2400 s-1 for the forward reaction and 230 s-1 for the back reaction. It is shown that the proposed new mechanism, based on transient kinetic measurements, also accounts for the results of previous investigations with other inhibitors (procaine, cocaine), which were carried out under both pre-steady-state and equilibrium conditions. A compound that binds to the same regulatory site on the receptor as MK-801 but does not affect the channel-opening equilibrium constant may have considerable use in protecting an organism from the effects of abused drugs.

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PROTEIN CONFORMATIONAL CHANGES IN THE μs TIME REGION INVESTIGATED WITH A LASER PULSE PHOTOLYSIS TECHNIQUE11We gratefully acknowledge financial support by grants (GM04942 and RR04804) awarded to G.P.H. by the National Institutes of Health.

Cited by 3 publications

References 27 publications

Mechanism of Glutamate Receptor-Channel Function in Rat Hippocampal Neurons Investigated Using the Laser-Pulse Photolysis (LaPP) Technique

Mechanism of Glutamate Receptor-Channel Function in Rat Hippocampal Neurons Investigated Using the Laser-Pulse Photolysis (LaPP) Technique

Inhibition of Nicotinic Acetylcholine Receptor by Philanthotoxin-343: Kinetic Investigations in the Microsecond Time Region Using a Laser-Pulse Photolysis Technique

On the Mechanism of Inhibition of the Nicotinic Acetylcholine Receptor by the Anticonvulsant MK-801 Investigated by Laser-Pulse Photolysis in the Microsecond-to-Millisecond Time Region

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