Photochemical Proteolysis of an Unstructured Linker of the GABA_AR Extracellular Domain Prevents GABA but Not Pentobarbital Activation

Abstract

The GABA type A receptor (GABA A R) is the major inhibitory receptor in the mammalian central nervous system and the target of numerous pharmaceuticals. The ␣-subunit of these pentameric Cys-loop neurotransmitter-gated ion channels contributes to the binding of both GABA and allosteric modulators such as the benzodiazepines, suggesting a role for this subunit in the conformational changes associated with activation of the receptor. Herein we use the nonsense suppression methodology to incorporate a photoactivatable unnatural amino acid and photochemically cleave the backbone of the ␣ subunit of the ␣ 1 ␤ 2 GABA A R in a linker region that is believed to span the subunit. Proteolytic cleavage impairs GABA but not pentobarbital activation, strongly suggesting that conformational changes involving this linker region are critical to the GABA activation pathway.

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

“…7C, Table 2), whereas insertions in the ␣-subunit were well tolerated. Consistent with the ␣Gly insertions being well tolerated, photochemical cleavage of the ␤4-␤5 linker in the GABA A R ␣-subunit does not affect PB EC 50 or its maximum current amplitudes (36). Regardless of the number of glycine insertions in either the ␤-subunits or the ␥-subunits, PB EC 50 increased about 10-fold ( Table 2), suggesting that GABA A R activation by PB does not depend on length and/or flexibility of the ␤-or ␥-subunit linkers.…”

“…These data are consistent with structural and molecular dynamic studies (1,3,4,(33)(34)(35) in the pLGIC family that indicate asymmetric subunit motions in the extracellular domain help power agonist-mediated gating. Recently, photochemical cleavage of the ␣-subunit GABA A R linker was shown to disrupt GABA activation (36). In this study, the cleavage site was located near the ␤/␣ interface close to loop E of the GABA binding site (at ␣M113, see Fig.…”

“…GABA A R Activation by PB Has Different Structural Requirements than GABA-Previous studies have shown that the structural elements underlying GABA A R activation by GABA and PB, which bind at different sites (25), are different (36,42,43). The differential effects of the glycine insertions on GABA and PB activation provide additional support for this idea.…”

The structural basis of function in Cys-loop receptors

“…In the heteromeric nAChR, this movement is initiated within the a-subunits, which undergo rotations, although several recent studies also describe movements that precede rotation (Horenstein et al 2001 ;Lape et al 2008;Pless & Lynch, 2009 ;Unwin et al 2002). The structural integrity of the ECD is important as weakening backbone hydrogen bonds in the b7, b9 and b10 sheets abolish receptor function, while photochemically cleaving the backbone between loops A and E has similar effects on GABA activation, but not on activation by pentobarbital (Gleitsman et al 2009 ;Hanek et al 2010). The movement of the ECD, mediated by the M2-M3 linker at the extracellular side of the TMD, destabilizes the hydrophobic ' girdle ' in the channel, which moves away from the centre of the pore into space that resides between the inner and outer rings, opening the channel (Law et al 2000 ;Miyazawa et al 2003).…”

In Vivo Incorporation of Non‐canonical Amino Acids by Using the Chemical Aminoacylation Strategy: A Broadly Applicable Mechanistic Tool

“…This residue has been incorporated into K + channels, nAChRs, [66] and the GABA A receptor, where it was used to probe the linkage between the orthosteric (GABA) binding site and the allosteric binding region that is the site of action of benzodiazepines. [67] A second-generation photochemical proteolysis residue based on decaging a selenide that then undergoes an intramolecular S N 2 reaction has also been developed. [68] …”