An Arginine Involved in GABA Binding and Unbinding But Not Gating of the GABA_AReceptor

Abstract

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 ␣ 1 ␤ 2 and ␣ 1 ␤ 2 -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.

GABA Affinity Shapes IPSCs in Thalamic Nuclei

“…These results differ from previous kinetic studies that find prominent fast and long-lasting desensitization components (Celentano and Wong, 1994;Bianchi et al, 2001;Wagner et al, 2004). The weak desensitization of GABA A Rs from thalamic neurons might be a nucleus-specific mechanism of functional significance.…”

“…GABA binds to the extracellular domain of the receptor complex at the interface between ␣ and ␤ subunits (Schofield et al, 1987;Amin and Weiss, 1993;Brejc et al, 2001). Sitedirected mutagenesis and molecular modeling have delineated the agonist binding site as formed by a series of ␤-sheet and loop motifs from ␣ and ␤ subunits, with several critical amino acid residues mediating molecular interactions between the receptor and agonist (Sigel et al, 1992;Wagner and Czajkowski, 2001;Wagner et al, 2004). Because the amino acid sequence is variable among isoforms, switching a single subunit (i.e., ␣1 to ␣3) can alter the identity of the key residues within the binding site (Bohme et al, 2004).…”

“…To examine the channel properties underlying synaptic and somatic patch receptors, nonstationary variance analyses of GABA A currents were performed (Sigworth, 1980;De Koninck and Mody, 1994;Wagner et al, 2004). Current events were sorted, and only events with rapid rise times (Ͻ2 ms) were included for analysis.…”

“…Among these, the rate of macroscopic desensitization is informative, because this parameter is shaped by the interplay of gating and desensitization time constants but not agonist binding or unbinding (Wagner et al, 2004). We examined receptor desensitization using sustained (1 s) applications of GABA to outside-out patches from the VB and RTN.…”

Kinetics and Spontaneous Open Probability Conferred by the ϵ Subunit of the GABAAReceptor

“…This estimate is lower than that reported previously (20 Ϯ 3%), using slower applications of GABA (100 M) and picrotoxin (1 mM) to oocytes (Mortensen et al, 2003). It is important to note that slow solution exchange biases peak current estimates toward lower values (Wagner et al, 2004), thus inflating the ratio of spontaneous current to peak GABA-evoked current.…”