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. 2009 Jun 26;284(26):17819-25.
doi: 10.1074/jbc.M900030200. Epub 2009 Apr 8.

A Lipid-Dependent Uncoupled Conformation of the Acetylcholine Receptor

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Free PMC article

A Lipid-Dependent Uncoupled Conformation of the Acetylcholine Receptor

Corrie J B daCosta et al. J Biol Chem. .
Free PMC article

Abstract

Lipids influence the ability of Cys-loop receptors to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. With the nicotinic acetylcholine receptor (nAChR) from Torpedo, current models suggest that lipids modulate the natural equilibrium between resting and desensitized conformations. We show that the lipid-inactivated nAChR is not desensitized, instead it adopts a novel conformation where the allosteric coupling between its neurotransmitter-binding sites and transmembrane pore is lost. The uncoupling is accompanied by an unmasking of previously buried residues, suggesting weakened association between structurally intact agonist-binding and transmembrane domains. These data combined with the extensive literature on Cys-loop receptor-lipid interactions suggest that the M4 transmembrane helix plays a key role as a lipid-sensor, translating bilayer properties into altered nAChR function.

Figures

FIGURE 1.
FIGURE 1.
Structure of the nAChR from Torpedo (Protein Data Bank code 2BG9), and a minimal model of nAChR conformational equilibria. A, the entire nAChR pentamer, with labeled extracellular agonist-binding (ABD), transmembrane pore (TMD), and cytoplasmic (CD) domains. Side chains of residues forming part of the agonist-binding site (1; αTrp-149) and the ion pore gate (2; αLeu-251, as well as analogous β-, γ-, and δ-subunit leucines) are shown in orange and purple. Views shown are from the synaptic space of B, the agonist-binding domain, and C, the transmembrane pore. D, our data show that lipid composition influences the activatable pool of receptors by controlling the proportion of nAChRs in uncoupled (U) versus coupled/resting (R) conformations (Scheme 1, boxed). Because gating appears to involve structural rearrangements at the lipid-protein interface (44), lipids may also influence the equilibrium between R, O (open), and D (desensitized) states (Scheme 2).
FIGURE 2.
FIGURE 2.
Influence of membrane lipid composition on nAChR-agonist interactions and conformational change. A, ±Carb difference spectra from native-nAChR (black), PC/PA/Chol-nAChR (blue), and PC-nAChR (red), with control spectra in gray (see supplemental Experimental Procedures). B, corresponding ±Carb difference spectra but in the presence of 200 μm dibucaine. The gray absorption spectrum (bottom) is aqueous dibucaine. Negative dibucaine vibrations result from Carb-induced displacement of dibucaine from the agonist-binding sites. Scale bars in both A and B represent 0.0001 (native) or 0.0005 (PC/PA/Chol and PC) absorbance units. C, Carb-induced changes in ethidium fluorescence for the same nAChR membranes. At the indicated times, 250 nm nAChR, 500 μm Carb, and 500 μm dibucaine (Dib) were added to a 0.3 μm ethidium solution. D, dibucaine displaceable fluorescence for nAChRs in each membrane environment in the presence (+) or absence (−) of 500 μm Carb (mean ± S.D., n = 9). E and F, the above difference spectra are calculated by subtracting a spectrum of state ”1“ from a spectrum of state ”2.“ G, schematic for the ethidium (Eth) fluorescence measurements. Ethidium fluoresces weakly in solution (left and right), but with greater intensity when bound to the desensitized nAChR pore (middle).
FIGURE 3.
FIGURE 3.
nAChR equilibrium binding affinities and allosteric coupling in different membrane environments. A, equilibrium binding of [3H]ACh to native-nAChR (gray), PC/PA/Chol-nAChR (blue), and PC-nAChR (red), normalized to the number of accessible agonist sites (see supplemental Experimental Procedures and Table S1). Each data point is the mean of three independent measurements (n = 3), with error bars in both the x and y direction representing ± S.D. B, changes in specific [3H]ACh binding as a function of TCP concentration for the nAChR in each membrane environment. All measurements were made with 100 nm [3H]ACh. Data points are mean ± S.D. (n = 3).
FIGURE 4.
FIGURE 4.
The effects of membrane lipid composition on nAChR thermal stability and solvent accessibility. A, proportion of nAChRs denatured as a function of increasing temperature, and B, fraction of unexchanged nAChR peptide hydrogens remaining after different lengths of time exposed to deuterated buffer. In each case, data were collected for PC/PA/Chol-nAChR (blue squares) and PC-nAChR (red circles), both in the presence (+; filled symbols) and absence (−; open symbols) of 500 μm Carb. The data in A are from single experiments that are representative of the means presented in Table S2. In B, after ∼0.25 h every fourth data point is shown (where error bars, mean ± S.D. are smaller than the data points; n = 6).
FIGURE 5.
FIGURE 5.
Potential role of M4 as a lipid-sensor modulating allosteric coupling in the Torpedo nAChR. A, view of the α-subunit agonist-binding domain (ABD) and transmembrane pore domain (TMD), highlighting structures at their interface (Cys-loop, post-M4, β1-β2 loop, and the M2-M3 linker). Also labeled are the C-loop and Trp-149, which form part of the agonist-binding site, and Leu-251 thought to form part of the pore gate. B and C, close up views of the coupling interface highlighting possible residues important for: B, relaying M4 (orange) induced pressure on the Cys-loop (green) to the M2-M3 linker (red); and C, M4 residues in close contact with the Cys-loop. D, schematic depicting possible lipid-dependent structural rearrangements of the transmembrane helices resulting in loss of interactions between the C-terminal end of M4 and the Cys-loop.

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