Molecular Dissection of Benzodiazepine Binding and Allosteric Coupling Using Chimeric γ-Aminobutyric Acid_AReceptor Subunits

Abstract

Although gamma-aminobutyric acid (GABA)A receptor alpha subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a gamma subunit is required for high affinity BZD effects. To determine which regions unique to the gamma2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat gamma2S and alpha1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevis oocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (chi) containing at least the amino-terminal 161 amino acids of gamma2S bound BZDs with wild-type affinity when coexpressed with alpha1 and beta2 subunits. Further analysis of the gamma2S binding site region uncovered two areas, gamma2S K41-W82 and gamma2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the gamma2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using gamma/alpha chimeras, we identified two gamma2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.

Structural Link between γ-Aminobutyric Acid Type A (GABAA) Receptor Agonist Binding Site and Inner β-Sheet Governs Channel Activation and Allosteric Drug Modulation

“…All mutant cDNAs were confirmed using double-stranded DNA. GABA A Rs were expressed in Xenopus laevis oocytes as described previously (19) and recorded from 2-14 days after injection.…”

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

“…Oocytes were prepared as described previously (Boileau et al, 1998). Capped cRNAs encoding the ␣ 1 , ␤ 2 , ␣ 1 K219C, ␣ 1 R220C, ␣ 1 K221C, ␤ 2 K213C, ␤ 2 K215C, and ␤ 2 R216C subunits in the vector pGH19 (Liman et al, 1992;Robertson et al, 1996) were transcribed in vitro using the mMessage mMachine T7 kit (Ambion, Austin, TX).…”

Altered kinetics and benzodiazepine sensitivity of a GABA A receptor subunit mutation [γ 2 (R43Q)] found in human epilepsy

“…binding interface with the ␣-subunit. Therefore, R43 is not likely to be part of the BZD-binding site (22). Modeling suggests further that ␥ 2 (R43) could form a salt bridge with ␥ 2 (E178), which may be involved in a second salt bridge across the subunit interface with ␤ 2 (R117) (29).…”

“…Xenopus oocytes (stage V or VI) were microinjected with 25 nl (Ϸ25 ng) of cRNA containing GABA A subunits in specific ratios. Because GABA A receptors can assemble as a binary complex of ␣ 1 and ␤ 2 alone, ␥ 2 was used in a 10-fold molar excess to promote assembly of ␣ 1 ␤ 2 ␥ 2 receptors (22). Oocytes then were stored in OR2 buffer (82.5 mM NaCl͞2 mM KCl͞1 mM MgCl 2 ͞5 mM Hepes, pH 7.5, supplemented with 2% FBS, 100 units͞ml penicillin, 100 g͞ml streptomycin; Life Technologies, Gaithersburg, MD) and incubated at 18°C for 3-5 days to allow for adequate receptor expression.…”