The energetics of neuromuscular acetylcholine receptor gating
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Nicotinic neuromuscular acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel family and function as signal transducers. The driving force for the opening conformational change is a ∼6000 fold increase in affinity for the neurotransmitter (acetylcholine) that involves rearrangements of atoms at each of the two transmitter binding sites. This energy is transmitted to the gate region in the pore that regulates ion permeation. In an attempt to increase our understanding of the mechanism of this reversible `gating' isomerization, we used single-channel electrophysiology and temperature to estimate gating rate and equilibrium constants and the associated energy and enthalpy changes. We investigated the temperature dependence of adult neuromuscular AChRs and observed that the differences in enthalpy of the affinity change for three agonists (acetylcholine, choline and tetramethyl ammonium) was approximately equal to the free energy differences. The affinity change is exothermic hence, the two binding sites are heat engines that drive the work of the gating conformational change. In wt AChRs, the agonist association and dissociation rate constants were approximately temperature insensitive, but in αG153S and choline they decreased markedly with temperature (Q 10 ∼7.4). The low-affinity agonist equilibrium dissociation constant did not change with temperature, so the enthalpy of the affinity change is specifically associated with the formation of the high-affinity nAChR complex. The free energy and enthalpy changes were found to be additive for a pair of mutations, implying that the principal energetic consequence of a mutation is independent and local. For 16 different mutations, the range of enthalpy change was greater than that of free energy because of compensating entropy changes. The side chain substitutions resulted in AChRs that were either heat or cold activated. &phis; (the correlation between the opening and gating equilibrium constants, on a log-log scale) was approximately constant with temperature both with and without agonists, indicating similar nAChR gating reaction pathways for these conditions. Regardless of the agonist and position of the residue, the activation enthalpy was approximately the same for channel opening and closing. Thus, the enthalpy of the gating barrier is not strongly influenced by perturbations such as agonists and mutations and it is possible to compute a thermal map of heat exchange in the nAChR gating isomerization. We found that loop 9 (in the extracellular domain) is mostly open-like in energy at the gating transition state (&phis;∼0.85) and that this structure regulates not only channel opening but also the stability of the intrasubunit-interface, assembly and expression of nAChRs. We also investigated the binding, gating equilibrium constants of mutations of two binding site residues in the complimentary subunit (εP123 and δP123 ProD2) that are conserved in all mammalian nAChRs. Both residues showed high diliganded phi value (∼0.97). Mutations in the ε subunit showed larger energy changes compared to that in the δ subunit. We were able to engineer nAChRs having only one functional transmitter binding site. We studied single-channel currents and quantified &phis; values for nAChRs with mutations in the pore lining, M2 helix of the δ subunit. The largest diliganded equilibrium constant changes were observed in the cytoplasmic half of δM2 (2', 5', 9' and 16'), with smaller changes apparent for residues ≥18'. &phis; was ∼0.32 for most δM2 residues suggesting that they experience late free-energy changes in the channel opening process.