Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor

Abstract

Benzodiazepines (BZDs) and barbiturates exert their CNS actions by binding to GABA-A receptors (GABARs). The structural mechanisms by which these drugs allosterically modulate GABAR function, to either enhance or inhibit GABA-gated current, are poorly understood. Here, we used the substituted cysteine accessibility method to examine and compare structural movements in the GABA-binding site interface triggered by a BZD positive (flurazepam), zero (flumazenil) and negative (3-carbomethoxy-4-ethyl-6,7-dimethoxy-β-carboline, DMCM) modulator as well as the barbiturate pentobarbital. Ten residues located throughout the GABA-binding site interface were individually mutated to cysteine. Wild-type and mutant α₁β₂γ₂ GABARs were expressed in Xenopus laevis oocytes and functionally characterized using two-electrode voltage clamp. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive methanethiosulfonate (MTS) reagents in the absence and presence of BZD-site ligands and pentobarbital. Flurazepam and DMCM each accelerated the rate of reaction at α₁R131C and slowed the rate of reaction at α₁E122C, whereas flumazenil had no effect indicating that simple occupation of the BZD binding site is not sufficient to cause movements near these positions. Therefore, BZD-induced movements at these residues are likely associated with the ability of the BZD to modulate GABAR function (BZD efficacy). Low, modulating concentrations of pentobarbital accelerated the rate of reaction at α₁S68C and β₂P206C, slowed the rate of reaction at α₁E122C and had no effect at α₁R131C. These findings indicate that pentobarbital and BZDs induce different movements in the receptor, providing evidence that the structural mechanisms underlying their allosteric modulation of GABAR function are distinct.

Introduction

GABA_A receptors (GABARs), besides being activated by the neurotransmitter GABA, are modulated by numerous therapeutically important drugs, including barbiturates, anesthetics, and benzodiazepines (BZDs). These compounds are allosteric modulators as they bind to sites distinct from the orthosteric GABA binding sites to potentiate or inhibit GABA-evoked current. Significant strides have been made in identifying the binding sites for these modulators (see (Hemmings et al., 2005, Mitchell et al., 2008, Sigel, 2002) for reviews). However, the structural mechanisms that couple the binding of these modulators to the allosteric modulation of GABA-gated current are less well understood.

BZDs have varying effects on GABAR function. BZD-site agonists (e.g. diazepam and flurazepam), act as positive modulators to enhance GABA-gated current (I_GABA). BZD-site inverse agonists (e.g. DMCM) act as negative modulators to inhibit I_GABA, while BZD-site antagonists (e.g. flumazenil) bind but have no effect on I_GABA and thus act as “zero” modulators. The BZD binding site is located in the extracellular amino terminal domain, at the interface between the GABAR α and γ subunits (Pritchett et al., 1989, Smith and Olsen, 1995). Previous studies have identified residues in Loop F/9, the M2–M3 extracellular loop and the pre-M1 region of the γ2 subunit that are required for enhancement of I_GABA by BZD positive modulators (Boileau and Czajkowski, 1999, Boileau et al., 1998, Hanson and Czajkowski, 2008, Jones-Davis et al., 2005, Padgett and Lummis, 2008). Interestingly, these γ2 subunit residues/regions are not critical for inhibition of I_GABA by the BZD-site negative modulator, DMCM (Boileau and Czajkowski, 1999, Hanson and Czajkowski, 2008), suggesting that the structural pathways mediating BZD negative allosteric modulation are distinct from those involved in positive modulation.

Depending on the electrophysiological approach and kinetic models used, investigators have asserted that BZDs exert their allosteric effects on I_GABA by either altering the microscopic binding of GABA at the orthosteric site (Goldschen-Ohm et al., 2010, Lavoie and Twyman, 1996, Mellor and Randall, 1997, Rogers et al., 1994, Thompson et al., 1999, Twyman et al., 1989), or by shifting the closed to open state channel equilibrium of the agonist-bound receptor (Campo-Soria et al., 2006, Downing et al., 2005, Rusch and Forman, 2005). Regardless of the mechanism, because GABA binding and channel gating are energetically coupled (i.e. the open channel state of the GABAR has a higher affinity for GABA than the closed state), one would predict that BZD actions would result in structural rearrangements of the GABA binding site.

Recently, we demonstrated that the GABA binding site undergoes structural rearrangement upon binding the positive BZD modulator flurazepam (Kloda and Czajkowski, 2007). Here, we tested whether positive, negative, and zero allosteric modulators trigger distinct conformational movements within the GABA-binding site interface that are correlated with their efficacy. We mutated ten residues throughout the GABA-binding site interface to cysteine (Fig. 1). We then measured the rate at which the introduced cysteines were modified by sulfhydryl-specific reagents in the absence and presence of flurazepam (BZD positive modulator), DMCM (BZD negative modulator), and flumazenil (BZD zero modulator) to probe the dynamics of the GABA binding interface upon binding BZD modulators that elicit different pharmacological effects. We also examined whether low concentrations of the positive allosteric modulator, pentobarbital, which binds at a site distinct from both the BZD and GABA binding sites (Amin, 1999, Belelli et al., 1999, Serafini et al., 2000), induces similar structural rearrangements at the GABA-binding site interface as a BZD positive modulator.