How fast does the gamma-aminobutyric acid receptor channel open? Kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.
The gamma-aminobuytric acid(A) (GABA(A)) receptor is a membrane-bound protein that mediates signal transmission between neurons through formation of chloride ion channels. GABA is the activating ligand, which upon binding to the receptor triggers channel opening in the microsecond time domain and reversible desensitization of the receptor in the millisecond time region. We have investigated the channel-opening mechanism for this receptor in rat hippocampal neurons before the protein desensitizes by using a rapid flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 30 micros time resolution to determine the rate and equilibrium constants for channel opening and closing. Two different forms of the receptor, namely, a rapidly and a slowly desensitizing form, exist in the rat hippocampal cells and are characterized by their different rates for desensitization. At 250 microM GABA the rate constant for desensitization was 2.3 +/- 0.4 s(-)(1) for the rapidly desensitizing form and 0.4 +/- 0.1 s(-)(1) for the slowly desensitizing form. The dissociation constant of GABA from the site controlling channel opening was 100 +/- 40 microM for the rapidly desensitizing form and 120 +/- 60 microM for the slowly desensitizing form. The rate constants for channel closing did not differ significantly for the two forms, 85 +/- 20 s(-)(1) for the rapidly desensitizing and 100 +/- 60 s(-)(1) for the slowly desensitizing form. However, the channel-opening rate constant differed by a factor of 3, 1840 +/- 160 s(-)(1) for the rapidly desensitizing and 6700 +/- 330 s(-)(1) for the slowly desensitizing form. This difference in the rate constant for channel opening for the two forms, determined by the laser-pulse photolysis technique, is reflected as a shift in the channel-opening equilibrium constant, which is 7 +/- 5 and 20 +/- 15 for the rapidly and slowly desensitizing forms respectively, determined by the cell-flow method. These constants, together with the concentration of GABA and the concentration of receptor sites in the membrane, determine the number of channels that open as a function of GABA concentration, and the rate at which they open and close. These constants play an important role in determining the rate of the transmembrane ion flux and, therefore, the receptor-controlled changes in transmembrane voltage that trigger signal transmission.
Jayaraman
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Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853-2703, USA.
Thiran
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Hess
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D000818:Animals; D006624:Hippocampus / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D007834:Lasers; D009474:Neurons / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D010782:Photolysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D018079:Receptors, GABA / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology
D007473:Ion Channels; D018079:Receptors, GABA