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The Journal of Neuroscience, March 17, 2004, 24(11):2733-2741; doi:10.1523/JNEUROSCI.4316-03.2004

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Cellular/Molecular
An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABA_A Receptor

David A. Wagner,¹ Cynthia Czajkowski,¹ and Mathew V. Jones¹

¹Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706

GABA_A receptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose–response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function.

Here, we focused on a residue (₂-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of ₂-R207, we expressed ₁₂ and ₁₂-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because ₂-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

Key words: kinetics; HEK293 cells; mutation; single channel; agonist; antagonist

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Received Sep 22, 2003; revised December 23, 2003; accepted December 24, 2003.

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