Inverse Effects on Gating and Modulation Caused by a Mutation in the M2-M3 Linker of the GABAA Receptor γ Subunit

Abstract

M2-M3 linkers are receptor subunit domains known to be critical for the normal function of cysteine-loop ligand-gated ion channels. Previous studies of ␣ and ␤ subunits of type "A" GABA receptors suggest that these linkers couple extracellular elements involved in GABA binding to the transmembrane segments that control the opening of the ion channel. To study the importance of the ␥ subunit M2-M3 linker, we examined the macroscopic and single-channel effects of an engineered ␥2(L287A) mutation on GABA activation and propofol modulation. In the macroscopic analysis, we found that the ␥2(L287A) mutation decreased GABA potency but increased the ability of propofol to enhance both GABA potency and efficacy compared with wild-type receptors. Indeed, although propofol had significant effects on GABA potency in wild-type receptors, we found that propofol produced no corresponding increase in GABA efficacy. At the single-channel level, mutant receptors showed a loss in the longest of three open-time components compared with wild-type receptors under GABA activation. Furthermore, propofol reduced the duration of one closed-time component, increased the duration of two open-time components, and generated a third open component with a longer lifetime in mutant compared with wild-type receptors. Taken together, we conclude that although the ␥ subunit is not required for the binding of GABA or propofol, the M2-M3 linker of this subunit plays a critical role in channel gating by GABA and allosteric modulation by propofol. Our results also suggest that in wild-type receptors, propofol exerts its enhancing effects by mechanisms extrinsic to channel gating.

A Single Phenylalanine Residue in the Main Intracellular Loop of α1 γ-Aminobutyric Acid Type A and Glycine Receptors Influences Their Sensitivity to Propofol

“…The absence of an effect of propofol on open time suggests that it does not affect the channel closing rate. Thus, the increased open probability observed in this study is either attributable to an increase in burst duration, which is in agreement with a study in GABA A Rs 40 , or to an increase in opening frequency. Further studies will be required to distinguish between these possibilities.…”

GABA Receptors: Pharmacological Potential and Pitfalls

“…Furthermore, the findings that propofol reduced desensitization and prolonged deactivation of receptors containing γ2L subunit and enhanced peak currents or prolonged deactivation of receptors containing δ subunit suggest that propofol-induced enhancement of both phasic and tonic inhibition may contribute to its anesthetic effect in the brain [145]. In accordance with mentioned studies, mutations in α and γ subunits change modulatory effects of propofol at the GABAA receptors [146,147], while particular residues in β subunits, distributed throughout the membrane-spanning region of the receptor, as well as in the extracellular domain, determine the propofol's actions. Thus, a point mutation in the β1 subunit (Met286Trp) abolished potentiation of GABA by propofol, but did not alter direct activation of the receptor by high concentrations of propofol [148].…”

De novo variants in GABRA2 and GABRA5 alter receptor function and contribute to early-onset epilepsy

“…Additionally, GABRB3 p.Y302C was previously identified in three unrelated patients with phenotypes that include focal epilepsy and intractable epileptic encephalopathy (Allen et al, 2013;Møller et al, 2017). The M2-M3 linker, where P301L and Y302C are located, is a highly conserved region that is known to be involved in coupling binding of the agonist to the gating of the channel, a function important for conferring ligand efficacy (O'Shea et al, 2009).…”