Borghese, C. M. | bioRxiv Science

bioRxiv2024

A single main-chain hydrogen bond required to keep GABAA receptors closed

GABAA receptors are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Despite significant progress understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified M2-M3 linker mutations that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the {beta}2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond during channel opening accounts for approximately one third of the activation energy derived from GABA binding. In contrast, the analogous H-bond in the 1 subunit has no effect on gating. Our observations suggest that channel opening involves state-dependent breakage/disruption of a specific main-chain H-bond within the {beta}2 subunit M2-M3 linker.

biophysics↗

bioRxiv2023

A potential cost of evolving epibatidine resistance in poison frogs

BackgroundSome poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) {beta}2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. ResultsThe introduction of S108C into the 4{beta}2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, 4{beta}2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested (Epipedobates anthonyi, non-dendrobatid frogs, as well as human), compared with 4{beta}2 nAChRs with the conserved S108. Immunolabeling of frog or human 4{beta}2 nAChRs in the plasma membrane using radiolabeled antibody against the {beta}2 nAChR subunit shows that C108 significantly decreased the number of cell-surface 4{beta}2 nAChRs, compared with S108. ConclusionsWhile S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted 4{beta}2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.

evolutionary biology↗