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 approximately equal to 48,000 s(-1) and approximately equal to 1,700 s(-1), respectively (in adult mouse, at 24 degrees 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 (tau(b)) elicited by a low concentration of the natural transmitter, acetylcholine. tau(b) increased in constructs with mutations that increased the gating equilibrium constant by either increasing the opening or decreasing the closing rate constant. However, tau(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 approximately 0.86 mus(-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.

Fig. 2.

Slow-closing and fast-opening mutants generate different burst-duration (τ_b) asymptotes. The x-axis values were calculated from the estimated gating rate constants, α_ACh and β_ACh, and the rate constant for ACh dissociation from closed AChRs, k_-2 (Eq. 3b). Each symbol is the mean of more than three patches. The asymptote of the slow-closing mutant series (○) is ≈2-fold greater than that of the fast-opening series (•), even though a simple kinetic model (Scheme 1 and Eq. 3) predicts that they should be the same. The specific mutations for each series (left to right) are given in sequence Table 1. The marked symbols are double mutations (Table 1). One possible explanation for the difference in the asymptote is that in the fast-opening series the opening rate constant reaches an upper boundary of ≈0.86 μs^-1.

Fig. 1.

Single-channel currents and burst-duration histograms for selected fast-opening and slow-closing AChR mutants. Currents were activated using 1 μM ACh. The durations are the time constants of the slowest burst-duration component (τ_b). (A) When the effect of the mutation is modest (see Table 1) there is little difference between fast-opening and slow-closing mutants. (B) When the effect of the mutation is large, the τ_b values for the fast-opening mutants are smaller than those for the slow-closing mutants. One possible reason for this difference is that AChRs can close arbitrarily slowly but cannot open arbitrarily fast.

Fig. 3.

Additional mutations do not increase the speed of channel opening. (Upper) Single-channel currents and burst-duration histograms for αS269I, a mutant that by itself opens with a rate that is near the speed limit. (Lower) αS269I on a background of αD97A, which by itself open at a rate that is near the speed limit. The τ_b values in both the single- and double-mutant constructs were similar, as expected given an upper-limit to the channel opening rate constant.