The molecular structure of the acetylcholine receptor

The electric organs of the electric ray Torpedo provide a rich source of acetyl-choline receptors. They can be isolated by using their specific binding to the snake venom a-bungarotoxin. The receptors are pentameric proteins with a total molecular weight of about 290 kDa. The subunits are called the a, ff, y and 8 chains; there are two a chains in each receptor and one of each of the others. The binding sites for acetylcholine are located on the a chains.

The acetylcholine receptor was the first ion channel to be sequenced by using recombinant DNA techniques. In 1982 the Kyoto University group (Noda and his colleagues) published the amino acid sequence of the a subunit, and sequences for the other subunits soon followed. The subunits varied in size from 437 amino acids (50 kDa) for the a chain to 501 amino acids (58 kDa) for the 8 chain.

The sequences for the four subunits show considerable homology. They all have four hydrophobic segments which probably form membrane-crossing helices (Fig. 7.10). The long section from the beginning of the chain to the first membrane-crossing helix is apparently all on the outside of the membrane. It contains disulphide crosslinks and sites for the attachment of sugars. In the a chain it contains the sites for binding acetylcholine and a-bungarotoxin.

How are the five subunits put together to form the whole complex? N. Unwin has used a sophisticated technique known as cryo-electron microscopy to answer this question. Nicotinic acetylcholine receptors can be isolated from Torpedo electric organ as regular close-packed arrays in tubular form. These arrays can be frozen and examined in the electron microscope, and the images subjected to Fourier analysis to give a three-dimensional map of the electron density in the molecule. Unwin was also able to examine the effects of acetylcholine on the structure by spraying it at the receptors just milliseconds before freezing them in liquid ethane at —178 °C.

The results of this procedure show that the five subunits form a receptor that has a large extracellular component to accommodate the two acetylcholine binding sites on the a subunits. The wide vestibule in the extracellular region narrows to a fine pore in the transmembrane region. At this narrow region the pore is lined by dense bent rods, which are probably the M2 segments of the five subunits. The upper part of Fig. 7.10 gives an impression of the whole structure.

In the resting state Unwin's model suggests that the channel is closed at its narrowest part by a large hydrophobic leucine residue. When acetylcholine binds to the a subunits, the M2 segments rotate somewhat to move these leucine residues out of the way so that cations can flow through the channel pore.

It is instructive to compare the nicotinic acetylcholine receptor channel with the voltage-gated channels described in Chapter 5. Unlike them, it is not gated by a change in membrane potential, and so, not surprisingly, there are no voltage sensors corresponding to the S4 segments of the voltage-gated channels. It belongs to a group of neurotransmitter-gated channels, which are themselves part of the general class of ligand-gated channels, which open when they bind a particular ligand molecule.

How can we be sure that the four subunits are sufficient to produce a functional receptor? The oocytes of the African clawed toad Xenopus have provided a most useful method for solving this problem.

Oocytes are large cells which are about to develop into mature eggs. They possess the normal translation machinery and so they will respond to the injection of messenger RNA by making the protein for which it codes.

Acetylcholine binding site

Synaptic cleft

Membrane

Cytoplasm

Acetylcholine binding site

Synaptic cleft

Membrane

Cytoplasm

Fig. 7.10. Diagrams of the molecular structure of the nicotinic acetylcholine receptor/channel. The complex consists of five subunits (upper diagram); the two a subunits contain acetylcholine binding sites. The amino acid chain of each subunit contains four membrane-crossing segments (lower diagram).

Fig. 7.10. Diagrams of the molecular structure of the nicotinic acetylcholine receptor/channel. The complex consists of five subunits (upper diagram); the two a subunits contain acetylcholine binding sites. The amino acid chain of each subunit contains four membrane-crossing segments (lower diagram).

Fig. 7.11. Expression of acetylcholine receptors in Xenopus oocytes. Messenger RNA from Torpedo electric organ is injected into an oocyte; two days later the oocyte membrane potential is voltage-clamped while acetylcholine is applied by ionophoresis (a). The record (b) shows the current response (upper trace) to acetylcholine; the lower trace monitors the ionophoresis current. From Barnard, Miledi and Sumikawa (1982).

Fig. 7.11. Expression of acetylcholine receptors in Xenopus oocytes. Messenger RNA from Torpedo electric organ is injected into an oocyte; two days later the oocyte membrane potential is voltage-clamped while acetylcholine is applied by ionophoresis (a). The record (b) shows the current response (upper trace) to acetylcholine; the lower trace monitors the ionophoresis current. From Barnard, Miledi and Sumikawa (1982).

Barnard and his colleagues injected oocytes with messenger RNA from Torpedo electric organ; two days later the oocyte would respond to application of acetylcholine by ionophoresis with a rapid depolarization, as is shown in Fig. 7.11.

Molecular cloning methods can be used to make messenger RNA coding for the different subunits of the acetylcholine receptor. Only when messenger RNAs for all four of the subunits were injected would the oocyte respond to application of acetylcholine. This shows that all four of the subunits are necessary for production of a functional receptor. It also shows that no extra components are required, so providing excellent confirmation for the conclusions of the recombinant DNA work.

Substances other than acetylcholine can combine with the receptors. Blocking agents such as a-bungarotoxin and curare combine with the receptors without opening their channels. Curare and some other compounds of this type are useful as muscular relaxing agents in surgery. Agonists of acetylcholine, such as nicotine and carbachol, combine with the receptors and do open the channels, so they induce ionic flow just as acetylcholine does.

In the disease myasthenia gravis it seems likely that the body produces antibodies to the neuromuscular acetylcholine receptor, resulting in partial neuromuscular block.

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