The Tetrameric Structure of a Glutamate Receptor Channel

Author:

Rosenmund Christian123,Stern-Bach Yael123,Stevens Charles F.123

Affiliation:

1. C. Rosenmund, Workgroup Cellular Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.

2. Y. Stern-Bach, The Hebrew University–Hadassah Dental School, Department of Anatomy and Embryology, Post Office Box 12272, Jerusalem, Israel.

3. C. F. Stevens, Howard Hughes Medical Institute, The Salk Institute, La Jolla, CA 92037, USA.

Abstract

The subunit stoichiometry of several ligand-gated ion channel receptors is still unknown. A counting method was developed to determine the number of subunits in one family of brain glutamate receptors. Successful application of this method in an HEK cell line provides evidence that ionotropic glutamate receptors share a tetrameric structure with the voltage-gated potassium channels. The average conductance of these channels depends on how many subunits are occupied by an agonist.

Publisher

American Association for the Advancement of Science (AAAS)

Subject

Multidisciplinary

Reference17 articles.

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3. Cloned Glutamate Receptors

4. After patch excision, we used a rapid perfusion system [C. Rosenmund, A. Feltz, G. L Westbrook, J. Neurosci. 15, 2788 (1995)] to produce rapid (200 to 400 μs) concentration changes at 0.1 to 0.2 Hz. The three-barreled squared capillary was filled with control, agonist, and antagonist. Switches were made from control → agonist → control and antagonist → agonist → antagonist. A version of this rapid switching method has been used previously to count binding sites [Clements J. D., Feltz A., Sahara Y., Westbrook G. L., J. Neurosci. 18, 119 (1998)]. We tried several agonists (for example, S-AMPA, glutamate), but quisqualate was most commonly used because it dissociated most slowly (half-decay times: AMPA = 64 ± 8 ms,n = 5; glutamate 6.2 ± 2.6 ms, n = 5; quisqualate = 106 ± 21 ms, n = 6). Pipettes were filled with 150 mM CsF, 20 mM HEPES, 2 mM MgCl2, 10 mM NaCl, 10 mM EGTA, and were adjusted to 320 mOsm and pH 7.3. The holding potential was –60 to –160 mV, and experiments were at room temperature. Currents were recorded with an Axopatch amplifier 200 B (Axon Instruments, Foster City, CA), low-pass filtered at 1 to 5 kHz, and digitized at 2 to 20 kHz. The extracellular medium contained 170 mM NaCl, 10 mM HEPES, 2 to 4 mM CaCl2, 2 to 4 mM MgCl2, and was adjusted to 330 mOsm and pH 7.25. Drugs were obtained from Tocris Cookson Ltd. (Bristol, UK) and dissolved in water- or dimethylsulfoxide-based stock solutions. Maximal final dimethylsulfoxide concentration was 0.1%.

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