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Neuropharmacology
2011 Jan 01;602-3:520-8. doi: 10.1016/j.neuropharm.2010.11.009.
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Allosteric modulators induce distinct movements at the GABA-binding site interface of the GABA-A receptor.
Sancar F
,
Czajkowski C
.
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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 α(1)β(2)γ(2) 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 α(1)R131C and slowed the rate of reaction at α(1)E122C, 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 α(1)S68C and β(2)P206C, slowed the rate of reaction at α(1)E122C and had no effect at α(1)R131C. 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.
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