Abstract
Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner β-sheet trigger channel activation. Homology modeling reveals that the β4-β5 linker physically connects the neurotransmitter binding site to the inner β-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor β-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.
Highlights
Structural elements and protein movements underlying GABAA receptor activation are not completely resolved
SR-95531 experiments were done at EC50 GABA for WT, ␣Gly, Gly[1], and ␥Gly insertions and at 30 mM GABA for Gly[2, 4], and 8 insertions. *, values are significantly different from WT, p Ͻ 0.05
Bars represent mean Ϯ S.E. of 10 –90% rise times from (n) experiments for WT and ␣Gly1␥ receptors. *, values are significantly different from WT, p Ͻ 0.05
Summary
Structural elements and protein movements underlying GABAA receptor activation are not completely resolved. Results: Glycine insertions in the extracellular 4-5 linker decrease GABA activation, invert antagonist efficacy, and reduce allosteric modulation. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner -sheet trigger channel activation. Inserting 1, 2, 4, and 8 glycines in this region of the GABAA receptor -subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the ␣- and ␥-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating
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