Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer

Abstract

The conformational changes underlying cysteine-loop receptor channel gating remain elusive and controversial. We previously developed a single-channel electrophysiological method that allows structural inferences about the transient open-channel conformation to be made from the effect and properties of introduced charges on systematically engineered ionizable amino acids. Here we have applied this methodology to the entire M1 and M3 segments of the muscle nicotinic acetylcholine receptor, two transmembrane alpha-helices that pack against the pore-lining M2 alpha-helix. Together with our previous results on M2, these data suggest that the pore dilation that underlies channel opening involves only a subtle rearrangement of these three transmembrane helices. Such a limited conformational change seems optimal to allow rapid closed-open interconversion rates, and hence a fast postsynaptic response upon neurotransmitter binding. Thus, this receptor-channel seems to have evolved to take full advantage of the steep dependence of ion- and water-conduction rates on pore diameter that is characteristic of model hydrophobic nanopores.

Figure 1. General properties of the AChR

(a) Membrane-threading pattern common to all Cysloop receptors, a superfamily of ligand-gated ion channels that, in vertebrates, includes receptors to acetylcholine, serotonin, γ-aminobutyric acid and glycine. The portion of the transmembrane domain studied in this paper is denoted in blue; in red, is the region studied in our previous work. (b) Ribbon representation of the transmembrane portion of the PDB file 2BG9 (ref. 1) a model of Torpedo's electric-organ AChR (a muscle-type AChR) in the closed state, as viewed from the extracellular side. The color code is the same as in a. In adult muscle, the γ subunit is replaced by the ε subunit. The molecular image was made with VMD (ref. 46). (c) Sequence alignment of the fifty-seven positions studied here. The four adult mouse-muscle AChR subunits (α1, β1, δ and ε) are compared. Vertical lines indicate the approximate ends of the α-helical stretches, largely as suggested by analysis of the PDB file 2BG9 with the program STRIDE (ref. 47). Positions that, according to our data, are oriented toward the lumen of the open-channel pore are indicated in bold for the four subunits.

Figure 2. Geometrical relationships between M1, M2, M3 and the pore in the closed state

(a) Distances from the Cα atoms to the long axis of the pore measured for M1 (○), M2 (•) and M3 (•). M1 and M3 residue numbers (in red and black, respectively) correspond to those of the δ subunit. M2 positions are denoted using the prime-numbering system (see Fig. 1a), which assigns positions α1Lys242, β1Lys253, δLys256 and εLys252 to 0′. (b) All possible Cα-Cα distances between the M1 segment of each subunit and the M2 segment of the subunit to the left (as seen from the extracellular side; Fig. 1b) were measured, and the shortest ones are plotted as a function of M1 residue number. The minima (indicated in red) identify the interfacial positions in M1. The interfacial positions in the (adjacent-subunit) M2 segment vary among subunits and are indicated next to these minima using the prime-numbering system. The mean value of the minima (that is, the mean Cα-Cα distance at the interface) is (10.8 ± 0.3) Å. (c) The mean Cα-Cα distance at the M3-M2 interface is (14.8 ± 1.1) Å. In this case, the “adjacent subunit” is the subunit to the right (that is, the one to which M3 is closest), as seen from the extracellular side (Fig. 1b). It is clear that M1 is closer to the adjacent subunit's M2 segment than M3 is. Means and standard errors were calculated from the values corresponding to the five individual subunits, and the unbroken lines are cubic-spline interpolations. All distances were measured on the PDB file 2BG9 (ref. 1) using VMD (ref. 46).

Figure 3. pH- and position-dependence of proton-transfer events

Individual protonationdeprotonation reactions of basic residues in cation-selective channels may be manifest in singlechannel patch-clamp recordings as fluctuations of the cation current between two levels of different conductance: a wild-type-like level (corresponding to the neutral, deprotonated side chain) and a level of lower conductance (corresponding to the positively-charged, protonated side chain). Arrows indicate the shut (zero current) and the two open-channel current levels. The shut level of each trace is also indicated by a horizontal broken line. Display f_c= 6 kHz. [ACh] = 1 μM. (a), (b), (c), (d) Example single-channel inward currents recorded from four M1 (a, b) and four M3 (c, d) mutants. The transmembrane potential in c and d is more hyperpolarized than that in a or b to better appreciate the difference between the two open-channel current levels.

Figure 4. The extent of channel block

(a) Current-voltage (I-V) relationships for some M1 and M3 mutants, and the wild-type AChR. For clarity, only the I-V curve corresponding to the blocked open state (that is, the “sublevel”) is shown for each mutant. Position 244 in the β1 subunit aligns with position 247 in the δ subunit. The I-V curves corresponding to two M2 lysine mutants, δL265K (9′ position, on the pore-facing stripe of M2) and δQ267K (11′ position, on the backside of M2) are also shown. To facilitate the visual comparison of slopes, the individual I-V curves were voltage-shifted so that they all go through the origin. (b) Extent-of-channelblock values calculated from the slopes of I-V curves for positions in and flanking M1 (○) and M3 (•). For comparison, the values corresponding to δM2 (ref. 11) are also shown (•). M1 and M3 residue numbers (in red and black, respectively) correspond to those of the δ subunit. δM1-M2 loop and δM2 positions are denoted using the prime-numbering system (the-7′ position corresponds to δPro250 whereas 25′ corresponds to δThr281; we omit the -3′ position in the δ subunit). The unbroken line is a cubic-spline interpolation. Proposed membrane boundaries are tentative. The close correspondence between these open-state findings and the closed-state structural model (Fig. 2a; remember that the ionizable groups project radially several ångströms out from the Cδ trace) implies that only minor conformational changes are associated with gating of the AChR. For most positions, the horizontal error bars (standard errors) are smaller than the symbols. The lumen of the pore would be to the right of the plot.

Figure 5. Two engineered lysines create two proton-binding sites

Example single-channel inward currents recorded at -100 mV from a construct having a lysine at position 225 of the α1 subunit (of which the muscle-type AChR has two copies). This position aligns with positions 239 in the δ subunit (Fig. 3a), 236 in the β1 subunit and 234 in the ε subunit. Arrows indicate the shut (zero current) and the three open-channel current levels. The shut level of each trace is also indicated by a horizontal broken line. Display f_c= 6 kHz. [ACh] = 1 μM. The rates and pK_as corresponding to the two proton-transfer reactions were estimated as described in Figure 6b, and are indicated in Table 1. As expected from the electrostatic effect of a neighboring positive charge, the proton-affinity of the monoprotonated AChR is lower than that of the deprotonated channel.

Figure 6. Experimental estimation of pK_a-values using electrophysiological recordings

The AChR interconverts among closed, desensitized (both referred here, collectively, as “shut states”) and open conformations with or without extra protons bound to the pore domain. The association and dissociation of single protons to and from the open state (but not to and from the shut states) may be manifest as discrete changes in the rate of ion flow. The three proton donors and three proton acceptors present in the solutions are indicated. BH and B⁻ denote the protonated and deprotonated forms of the H⁺-buffer, respectively. Note that the kinetics of both proton transfer and channel shutting affect the duration of sojourns in the different open-channel current levels (unbroken arrows). (a) Basic kinetic scheme used to estimate transition rates of mutants bearing a single engineered basic residue. (b) Basic kinetic scheme used in the case of the α1-subunit double-mutant constructs, which contain two lysines (one in each copy of the α1 subunit). This model assumes that both engineered lysines behave identically as far as the kinetics of proton association and dissociation are concerned, and allows for interactions between them. Models that allow the two proton-binding sites to be different did not yield consistent results. Rates and pK_a-values in Table 1 and Supplementary Table 1 are expressed per protonation site; that is, they were corrected for the statistical factor.

Figure 7. pK_a-shifts at protein-protein interfaces

Lysine side-chain ΔpK_a-values mapped onto ideal α-helical wheel representations of eighteen-residue, membrane-embedded stretches of M1 (a) and M3 (b) of the δ subunit. The size of the symbols increases toward the extracellular end. Wild-type residues, and the C and N termini are indicated. The color code is the same for both a and b. The lumen of the pore would be to the right of each plot. A thick black edge on the symbols identifies the positions in the α1, β1, δ and/or γ subunits of Torpedo's AChR that incorporate hydrophobic photoaffinity labels in the closed state in a manner that is consistent with the label reacting from the lipid bilayer,. Symbols with a grey edge indicate the positions in M1 that lie at the interface between M1 and M2 (of the same subunit) whereas those with a dark red edge indicate the positions in M3 at the intrasubunit M3-M2 interface, as suggested by the contact plots in Supplementary Figure 1. The δV307K mutation, in M3, prevented the expression of functional AChRs.