An Epilepsy-Related Region in the GABA_A Receptor Mediates Long-Distance Effects on GABA and Benzodiazepine Binding Sites

Abstract

The GABA A receptor mutation ␥ 2 R43Q causes absence epilepsy in humans. Homology modeling suggests that ␥ 2 Arg43, ␥ 2 Glu178, and ␤ 2 Arg117 participate in a salt-bridge network linking the ␥ 2 and ␤ 2 subunits. Here we show that several mutations at these locations exert similar long-distance effects on other intersubunit interfaces involved in GABA and benzodiazepine binding. These mutations alter GABA-evoked receptor kinetics by slowing deactivation, enhancing desensitization, or both. Kinetic modeling and nonstationary noise analysis for ␥ 2 R43Q reveal that these effects are due to slowed GABA unbinding and slowed recovery from desensitization. Both ␥ 2 R43Q and ␤ 2 R117K also speed diazepam dissociation from the receptor's benzodiazepine binding interface, as assayed by the rate of decay of diazepam-induced potentiation of GABAevoked currents. These data demonstrate that ␥ 2 Arg43 and ␤ 2 Arg117 similarly regulate the stability of both the GABA and benzodiazepine binding sites at the distant ␤/␣ and ␣/␥ intersubunit interfaces, respectively. A simple explanation for these results is that ␥ 2 Arg43 and ␤ 2 Arg117 participate in interactions between the ␥ 2 and ␤ 2 subunits, disruptions of which alter the neighboring intersubunit binding sites in a similar fashion. In addition, ␥ 2 Arg43 and ␥ 2 Glu178 regulate desensitization, probably mediated within the transmembrane domains near the pore. Therefore, mutations at the ␥/␤ intersubunit interface have specific long-distance effects that are propagated widely throughout the GABA A receptor protein.

A State-Dependent Salt-Bridge Interaction Exists across the β/α Intersubunit Interface of the GABAAReceptor

“…We used a simplified model of GABA A receptor behavior ( Fig. 6) that had been described previously (Jones et al, 1998;Wagner et al, 2004;Goldschen-Ohm et al, 2010). Although we considered more complex variations of this model that included additional desensitized and open states, the simplified model was equally suitable to recapitulate our data.…”

“…Although we considered more complex variations of this model that included additional desensitized and open states, the simplified model was equally suitable to recapitulate our data. During optimization the rate constant k on and P o-max were constrained to the value obtained from experiments in this study; all other parameters were initially set to values reported by Goldschen-Ohm et al (2010) and were unconstrained. Current responses from 3-and 500-ms pulses of saturating GABA were simultaneously fit for each patch.…”

“…Nonstationary variance analysis (Sigworth, 1980) was performed on responses to repeated 3-ms pulses of saturating GABA. As described previously (Wagner et al, 2004;Goldschen-Ohm et al, 2010), mean current (I) and variance ( 2 ) of the repeated pulses were calculated at each time point, mean current was divided into 100 equally sized bins, and the variances in each bin were averaged. The binned variance was plotted versus current and fit with the equation:…”

Different Residues in the GABAAReceptor Benzodiazepine Binding Pocket Mediate Benzodiazepine Efficacy and Binding

“…In general, it is believed that BZDs exert their allosteric effects by either shifting the GABA A R closed to open-state channel equilibrium (Downing et al, 2005;Rü sch and Forman, 2005;Campo-Soria et al, 2006) or altering the receptor's microscopic binding affinity for GABA (Twyman et al, 1989;Rogers et al, 1994;Lavoie and Twyman, 1996;Mellor and Randall, 1997;Thompson et al, 1999;Goldschen-Ohm et al, 2010). Regardless of the mechanism, BZD binding to the receptor is the initial perturbation that triggers structural rearrangements in the protein that result in modulation of GABA A R function.…”

Three Arginines in the GABAAReceptor Binding Pocket Have Distinct Roles in the Formation and Stability of Agonist- versus Antagonist-Bound Complexes

“…Unlike the binding rate, we could not examine this directly, but instead we asked whether such a hypothesis could explain our observations using a kinetic model shown previously to account for multiple aspects of GABA A receptor behavior (Fig. 7A) (Jones et al, 1998;Wagner et al, 2004;Goldschen-Ohm et al, 2010).…”