A speed limit for conformational change of an allosteric membrane protein

Abstract

Neuromuscular acetylcholine receptors are synaptic ion channels that open and close with rate constants of Ϸ48,000 s ؊1 and Ϸ1,700 s ؊1 , respectively (in adult mouse, at 24°C, ؊100 mV membrane potential). Perturbations of many different sites in the protein can change these rate constants, with those in the extracellular domain mainly affecting channel-opening and many of those in the membrane and intracellular domains mainly affecting channel-closing. We used single-channel recordings to measure the total open time per activation ( b) elicited by a low concentration of the natural transmitter, acetylcholine. b increased in constructs with mutations that increased the gating equilibrium constant by either increasing the opening or decreasing the closing rate constant. However, b did not approach the same asymptote in fast-opening and slow-closing constructs. The maximum value for the slow closers was about twice that for the fast openers. One interpretation of this difference is that there is an upper limit to the channel-opening rate constant, which we estimate to be Ϸ0.86 s ؊1 . One possibility is that this limit is the rate of conformational change in the absence of an overall activation barrier and thus reflects the kinetic prefactor for the acetylcholine receptor opening isomerization.channel gating ͉ nicotinic acetylcholine receptor ͉ energy landscape ͉ transition state A t cholinergic synapses, transmitter molecules trigger acetylcholine (ACh) receptor channels (AChRs) to switch from a stable conformation in which ion permeation is forbidden (''closed'') to one in which cations can permeate rapidly (''open''). This reversible change in structure, known as gating, involves the organized movements of many of the Ͼ2,300 residues within the five subunits that comprise this allosteric membrane protein (1-3). The driving energy for the reaction is a change in binding energy for the ligand; thus, gating must, at least, entail movements of residues at the two transmitter binding sites and in the ion conduction pathway.When activated by the natural transmitter ACh, mouse neuromuscular AChRs open rapidly (Ϸ48,000 s Ϫ1 ) and close relatively slowly (Ϸ1,700 s Ϫ1 ) (at 24°C, Ϫ100-mV membrane potential) (4). In single-molecule patch-clamp recordings with a time resolution of Ϸ25 s, the closed^open isomerization appears to be a two-state reaction with no directly detectable intermediate states. However, these states can be probed indirectly by using rate-equilibrium free energy (REFER) analyses (5-7). For a series of point mutations, the slope (⌽) of a log-log plot of the opening rate constant vs. the equilibrium constant is an index of the extent of progress of the perturbed site at the transition state of the reaction. In fully liganded AChRs, there is, to a first approximation, a longitudinal gradient in ⌽-values, with residues near the transmitter binding sites being open-like (⌽ Ϸ 1) and some of those in the transmembrane region being closed-like (⌽ Ϸ 0) at the transition state (8, 9). These results have been inte...

Seeking Structural Specificity: Direct Modulation of Pentameric Ligand-Gated Ion Channels by Alcohols and General Anesthetics

“…This structure introduced new possibilities for detailed homology modeling; for example, it was used to build both glycine and GABA A receptor structures (Kash et al, 2003;Trudell and Bertaccini, 2004) in an effort to understand how the binding energy of a ligand was coupled to an ion channel gating motion 40 Å away (Chakrapani and Auerbach, 2005). Recent NMR structures of isolated nicotinic receptor transmembrane domains (Bondarenko et al, 2012) further clarified the topological map and informed its use as a modeling template.…”

“…However, spontaneous gating occurs infrequently, and when it does, the transition takes approximately 20-100 microseconds (Chakrapani and Auerbach, 2005). Because it is difficult to simulate these time scales using molecular dynamics, normal mode analysis has been used to look at long time-scale collective motions (Bertaccini et al, 2005).…”

Energy landscape of the reactions governing the Na + deeply occluded state of the Na + /K + -ATPase in the giant axon of the Humboldt squid

“…The value of the transition entropy, ΔS ‡ ¼ 89.8 − R ln A, necessarily requires a knowledge of the factor A, which for large molecules in aqueous solutions is largely unknown. For some protein conformational changes involved in protein folding or ion channel opening, however, experimental estimates have put the value of A at approximately 10 6 s −1 (34,35). With this assumption, ΔS ‡ is approximately 62 cal∕molK.…”

“…where ΔH ≠ and ΔS ≠ correspond to the activation enthalpies and entropies for the deocclusion (subindices 4) and occlusion (subindices 3), respectively, A is the preexponential term, which has been set to be 10 6 s −1 (34,35) and assumed to be the same for the slow deocclusion and occlusion reactions, C 0 is the standard state concentration of 1 mol∕L (¼1∕1661 Å 3 ; see ref. 36), and ΔH u and ΔS u are the total enthalpy and entropy, respectively, of the adjacent binding reaction (binding of the third Na þ and unbinding of the first Na þ released).…”

Quaternary Speeding in Hemoglobin

“…A time constant of only 1.2 μs has been reported for ion channel opening in the multimeric membrane protein AChR. 1 And in this issue of JMB, Cammarata et al 2 report 2 μs as the time constant for the R-T quaternary transition in hemoglobin (Hb), whereas 20 μs had long been the accepted value. 3 Gibson's classic study of the recombination kinetics following photodissociation of the CO adduct, HbCO, revealed fast and slow phases with a 20-μs transition between them.…”