Linking the Acetylcholine Receptor-Channel Agonist-Binding Sites with the Gate

Abstract

The gating isomerization of neuromuscular acetylcholine receptors links the rearrangements of atoms at two transmitter-binding sites with those at a distant gate region in the pore. To explore the mechanism of this reversible process, we estimated the gating rate and equilibrium constants for receptors with point mutations of alpha-subunit residues located between the binding sites and the membrane domain (N95, A96, Y127, and I49). The maximum energy change caused by a side-chain substitution at alphaA96 was huge (approximately 8.6 kcal/mol, the largest value measured so far for any alpha-subunit amino acid). A Phi-value analysis suggests that alphaA96 experiences its change in energy (structure) approximately synchronously with residues alphaY127 and alphaI49, but after the agonist molecule and other residues in loop A. Double mutant-cycle experiments show that the energy changes at alphaA96 are strongly coupled with those of alphaY127 and alphaI49. We identify a column of mutation-sensitive residues in the alpha-subunit that may be a pathway for energy transfer through the extracellular domain in the gating isomerization.

Correlating Structural and Energetic Changes in Glycine Receptor Activation

“…5). The ␣1 Ϫ26Ј -␣1 19Ј coupling energy is well within the range for a significant interaction between residues in pLGICs during activation (39,40,44) and between ligand binding residues and ligand (45). We propose that this interaction represents one point of energy transfer along what is likely to be a broad reaction coordinate (broad corrugated activation barrier) as proposed by Auerbach for the related nAChR (40,46).…”

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

“…Mutation of either of these residues alters GABA-mediated current responses, suggesting that this salt bridge is important for linking the binding site to loop 2 in the gating interface (41). Consistent with this idea, recent work from Cadugan and Auerbach (16) has shown that ␣A96 (loop A) and ␣I49 (loop 2) in the mouse nAChR, which are aligned to ␤K102 and ␤D56 of the GABA A R, respectively, are energetically coupled.…”

“…1) and is in an ideal position to propagate initial binding site movements to gating movements in the channel domain. Consistent with this idea, molecular dynamic simulations of the nAChR identified motions in the ␤4-␤5 linker that were correlated with motions in loops 2, 7, and M2-M3 near the extracellular mouth of the channel (14), and mutations in the nAChR ␣-subunit ␤4-␤5 linker alter channel gating (15,16). Based on the crystal structures of the nAChR ␣-subunit extracellular domain, AChBP, GLIC, ELIC, and the invertebrate glutamate-activated chloride channel (2,3,(5)(6)(7)(8), this linker region spans each subunit and is relatively unstructured.…”

“…3B), suggesting that ␤Gly1 alters channel gating by decreasing the channel opening rate. In the GABA A R, nAChR, and serotonin type-3 receptor, mutating residues in the ␤4-␤5 linker near loop A increase unliganded channel opening (15,16,37,38), indicating that this region influences channel gating. Moreover, agonist-mediated movements in the nAChR ␣-subunit ␤4-␤5 linker have been observed (39).…”

Subunit Symmetry at the Extracellular Domain-Transmembrane Domain Interface in Acetylcholine Receptor Channel Gating

“…The Ala and Asn substitutions had the largest effect and decreased E 2 by ϳ180-fold. The range energy for ⑀Arg 3Ј was ϳ3.1 kcal/mol, which is about the median value for two ␣-subunit mutations (10,30). The R/E plot for ⑀Arg 3Ј had a slope (⌽-value) of 0.73 Ϯ 0.06 (Fig.…”