Signal Transduction
M1–M4: an unfortunate nomenclature, easily confused with the terminology for the muscarinic receptors: M1–m5, etc.
Fig 3.7 Structure of the nicotinic receptor without ligand.
Side and top views of the pentameric structure are shown in the central figures. One of the -subunits
is redrawn on the left to show its secondary structure. The images on the right indicate the M2 helices (red) that line the channel (2bg9.16).
Architecture of the nicotinic receptor
Because it is difficult to grow crystals of transmembrane proteins, the three-dimensional structure of the nicotinic receptor has been studied by cryoelectron microscopy of helical tubes prepared from the receptorrich postsynaptic membranes of Torpedo marmorata.15 Although electron microscopy provides a low to medium resolution approach, applying crystallographic refinement methods to the data has yielded a structure having atomic resolution (4 Å).16 This is shown in Figure 3.7. The receptor is pentameric and its five subunits, two of which are identical, are arranged
around a central pore. The clockwise arrangement viewed from the synapse is , , , , . All five subunits must be present for the complex to function as a channel. The overall shape of the receptor is a hollow cone, approximately 80 Å in diameter at its widest point and some 160 Å long (i.e. 8 16 nm). Its position with respect to the plasma membrane is indicated in Figure 3.7. The assemblage protrudes about 80 Å into the synaptic space and within this protuberance there is a central vestibule 20 Å in diameter. On the intracellular side of the membrane, the structure protrudes about 20 Å
and it contains another, smaller cavity with side openings. The two cavities communicate through the central pore.
The sequences of the individual subunits indicate extensive homology: 35–40% amino acid identity, with many additional conservative substitutions. The four
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Receptors
separate polypeptides evolved from a common ancestor by a process of successive gene duplication and modification that has its origin more than 1.5 109 years ago (thus long preceding the emergence of metazoan creatures). Before the three-dimensional structure of the nicotinic receptor was obtained, it was known from the sequences that each subunit possesses four stretches, M1, M2, M3, and M4, in which hydrophobic amino acids predominate (Figure 3.8), suggesting that these regions are membrane-spanning chains. Organized as-helices, their lengths would be sufficient to traverse the lipid bilayer.
Subunit structure
The prediction that the M1–M4 sequences of each of the subunits are indeed transmembrane -helices is confirmed by the structural data (Figure 3.7). The even number of traverses (4) means that the N- and C-terminals must be on the same side of the membrane. In fact they are extracellular. The exposed N-
terminal portion of each subunit is glycosylated and consists of some 200 amino acids assembled around a 10-stranded -sandwich, plus a short -helix (Figure 3.7). For the -subunit, loops connecting the -strands on one side of the molecule are involved in ligand binding. These are the A, B, and C loops, the Cysloop and the 1– 2 loop (not shown). The remaining residues that contribute
to the binding site are provided by the adjacent subunits ( or ). Both binding sites must be occupied in order to activate the receptor. The inhibitor of the channel, -bungarotoxin, binds in the close vicinity of these sites.
The loops linking the transmembrane spans, M1–M2 on the inside of the cell and M2–M3 on the outside, are relatively short, but the M3–M4 loop that
Such stretches of about 20 amino acids, having higher than average hydrophobicity, are present in all known membrane-spanning proteins. Furthermore, in those proteins
for which structural information is available, e.g. bacteriorhodopsin, the photosynthetic reaction centre of
the purple bacterium
Rhodopseudomonas,17 the potassium channel of Streptomyces lividans,18 and a gated mechanosensitive ion
channel in Mycobacterium tuberculosis,19 the hydrophobic spanning segments have all been shown to be organized as-helices.
Fig 3.8 Hydropathy plot of a nicotinic receptor -subunit.
The graph represents the hydropathy profile of the -subunit of the nicotinic acetylcholine receptor. The diagram depicts the transmembrane chain organization. The hydrophobicity index is the average hydrophobicity of stretches of 19 amino acids centred on each position in the chain (thus each point represents the average hydrophobicity of 9 1 9 amino acids). Each amino acid is assigned a hydrophobicity value as indicated in Figure 3.9.
Based on data for the human 7 subunit published in the LGIC database (www.pasteur.fr/recherche/banques/LGIC/LGIC.html).20
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Signal Transduction
A photoaffinity label is a compound that becomes reactive when subjected to a pulse of UV light. This enables it to form stable derivatives with molecules in its very close vicinity
forms the intracellular structure, is more extended (about 130 amino acids) and contains the intracellular MA helix. The non-helical part of this loop is unstructured in the EM data and is therefore omitted from Figure 3.7.
M2 sequences and the pore
Inspection of the sequences of the M2 regions of each of the subunits illustrates their homology (Figure 3.9). Within these short stretches there are at least eight points of identity and ten more that are conservative
substitutions. Notice also that each of these four sequences contains a dozen or more strongly hydrophobic amino acids (leucine, valine, isoleucine, etc., with hydrophobicity assignments 0).
There is much evidence that residues present in the M2 helices of all five subunits are the main contributors to the pore along the central axis of symmetry of the pentameric structure. Importantly, only residues of the M2 segments bind watersoluble channel-blocking agents, such as chlorpromazine and other compounds having photoaffinity properties. The rate of binding of these compounds is
1000 times greater when the channel is in its open configuration.22 In addition, fractionation of membrane proteins following photoaffinity labelling with lipid-soluble probes has shown that the M2 chains are fully shielded from the lipid environment of the membrane. Again, structural studies confirm that the transmembrane pore is lined by the M2 helices of each of the five subunits and
Fig 3.9 M2 sequences of the four subunits of the nicotinic receptor.
(a) M2 sequence comparison (Torpedo marmorata) indicating identical and conserved residues, the transmembrane region and the probable location of the channel gate. The -subunit residues in red are those that have pore-facing side chains. (b) Hydrophobicity values
assigned to the 20 protein amino acids. In (a) the hydrophobicities of the M2 residues are indicated in colours that correspond to the assignments shown in (b). (These assignments were calculated from data including vapour pressures and partition coefficients by Kyte and Doolittle.21)
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Receptors
show that this assembly is surrounded by an outer ring of helices (M1, M3, M4 from each subunit), that shields it from the plasma membrane, as depicted in Figure 3.7. The rather limited number of contacts between the outer shell and the M2 chains allows the movement necessary for pore opening to occur.
By probing the open channel with chlorpromazine or other site-specific channel-blocking substances, it has been possible to map its lining. Collectively, these experiments have identified amino acids separated by three or four residues along this section of the chain, consistent with an-helical arrangement. As the pore tapers to its smallest diameter, within the transmembrane region, the M2 segments are aligned so that the homologous residues form rings that are mostly non-polar (Figure 3.9). Polar or charged side chains mark the ends of the transmembrane region and the negatively charged groups will be selective for cations. At the narrowest point, the encircling hydrophobic side chains make contacts across the opening, keeping it effectively shut (an aperture 6–7 Å across, too small to admit a Na or K ion with its primary hydration shell, 8 Å in diameter). The pore region opens out on either side of the membrane into the vestibules within the structure. The lining of the vestibules and the surfaces of the openings in
the intracellular assembly, possess areas of negative charge. It is probable that these, as well as the anionic side chains exposed in the vicinity of the pore, contribute to the cationic selectivity of the channel.
Ligand binding and channel opening
Although the four different subunits ( , , , ) that constitute the nicotinic receptor are very similar, within the pentameric structure the two -subunits are organized differently from the others. This is due to a difference in the arrangement of their inner -sheets, resulting in an anticlockwise rotation ( 10° as viewed from outside the cell) relative to the non- -subunits. This has been termed a ‘distorted’ conformation.16 Relief of this distortion is thought to lead to channel opening. Understanding the activation mechanism has been advanced by comparing the ligand-free receptor with the watersoluble acetylcholine-binding protein of the snail Limnea stagnalis. This is homologous to the extracellular domain of the receptor. Figure 3.10 shows part of its structure with a ligand molecule (carbamylcholine) bound between two adjacent subunits.23 The loops that wrap around the ligand and that contribute to the binding site are shown and these correspond to the ligandbinding loops (A, B, C) indicated in Figure 3.7. (Note: unlike the nicotinic receptor, the acetylcholine-binding protein is a homopentamer of five 7 subunits). A comparison of the two structures suggests that ligand binding to a receptor relieves the twisted conformation of the -subunits and this movement is then communicated through the -strands to the M2 helices, so perturbing the hydrophobic interactions across the gate region and allowing it to open.
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