Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels: a single-channel study of second transmembrane segment 12' mutants

Abstract

The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12' position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C <--> O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the delta12' residue makes a more pronounced contribution to unliganded gating than the homologous residues of the alpha, beta, and straightepsilon subunits; (c) combinations of second transmembrane segment 12' mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at delta12' is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (alphaY93F, alphaY190W, and alphaD200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12' mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio.

Figure 1

Wild-type unliganded gating. (A) Continuous single-channel traces of spontaneous openings recorded at an estimated membrane potential of −120 mV. For display purposes, f_c ≅ 6 kHz. Openings are downward deflections. (B) Schematic representation of the residues occupying the M2 12′ position in wild-type receptors. The ordering of the subunits, as seen from the extracellular side, is as suggested by Machold et al. 1995b.

Figure 3

Set of reaction schemes used for kinetic modeling according to a full-likelihood approach. C, nonconductive; O, open. Whether these models represent different closed–open transitions of individual receptors or the gating activity of heterogeneous receptors in the patch is not known.

Figure 2

δS→T AChR unliganded gating. (A) Continuous single-channel traces of spontaneous openings recorded at approximately −100 mV. Note the multiplicity of closed- and open- time components. Display f_c ≅ 6 kHz. Openings are downwards. (B) Schematic representation of the residues occupying the M2 12′ position in δS→T receptors. (C) I-V curve under cell-attached conditions and least-square fit with a straight line.

Figure 6

Voltage dependence of 12′ δS→T AChR unliganded gating. Solid lines are fits to the Boltzmann equation for voltage dependence. Because only the brief type of openings was recorded in this patch, the full-likelihood approach was applied to a C ↔ O scheme. The opening rate is assumed to be the product of the opening rate constant and the number of channels in the patch. The particular value of the latter does not affect the zδ estimate.

Figure 4

Dwell-time histograms and superimposed density functions corresponding to wild-type and M2 12′ mutant AChR unliganded gating. The kinetics of δS→T, δS→T + βT→S, and εT→P receptors were fitted to Fig. 3 A. Those of the wild-type and δS→T + εT→S, δS→A, δS→N, δS→I, δS→P, and βT→P receptors were fitted to Fig. 3 B. Those of δS→Y and αT→P receptors were fitted to Fig. 3 G. The corresponding rate constant estimates are listed in Table . Each histogram corresponds to data recorded in one example patch for each construct.

Figure 5

Example bursts of unliganded openings of wild-type and M2 12′ mutant receptors. Display f_c ≅ 6 kHz. The different current amplitudes most likely reflect differences in the membrane potential. This is most evident in the case of the εT→P mutant, where a somewhat detached patch could have decreased the cell's membrane potential. Openings are downwards.

Figure 7

Effect of binding-site mutations on unliganded gating. (A) Representative single-channel traces in the absence of ligand. Display f_c ≅ 6 kHz. Openings are downwards. The background receptor on which the binding-site mutations were engineered was the double mutant 12′ δS→T + εT→S. (B) Bar representation of opening frequencies, mean open times, and the corresponding standard errors for the four constructs. The number of analyzed patches in each case is indicated in parentheses. The constructs having the binding-site mutations displayed only one type (the briefest one) of openings. However, the kinetics of the 12′ δS→T + εT→S mutant alone were best described by two open states (Fig. 3 B) with mean open times of 89.7 μs (88% of the total openings) and 4.66 ms (n = 4). In this case, only the rates associated with the briefest component were considered for comparison with the other three constructs. While the binding-site mutations greatly reduced the frequency of brief openings, little effect was exerted on their mean duration. “Opening frequencies” are the opening rates. Mean open times were calculated as the reciprocal of the closing rate constants, and the corresponding standard errors were calculated using and 2 in the companion paper.

Figure 8

Effect of the 12′ δS→T mutation on diliganded activity. Display f_c ≅ 6 kHz. In addition to inward currents (approximately −100 mV), outward currents (approximately +50 mV) were also recorded in the presence of 2 mM ACh or 2 mM acetylthiocholine to relieve fast blockade by the agonist itself. This was not necessary in the presence of 20 mM choline because the increase in open-channel noise due to fast blockade was much lower (Grosman and Auerbach 2000). Openings are downward deflections at −100 mV and upward deflections at +50 mV. The higher amplitude of the δS→T currents in the presence of 2 mM ACh at +50 mV, as compared with that of the wild type, is most likely due to the slower closing rate constant of the mutant.