All Five Subunits in the Nicotinic Acetylcholine Receptor Contribute to the Ion Channel

The acetylcholine receptor from skeletal muscle is a pen-tameric protein with a subunit composition of a2p^8. The a, P, 7, and 8 subunits have considerable sequence homology; on average, about 35-40 percent of the residues in any two subunits are similar. The complete receptor has fivefold symmetry, and the actual cation channel is a tapered central pore lined by homologous segments from each of the five subunits (Figure 7-45).

The channel opens when the receptor cooperatively binds two acetylcholine molecules to sites located at the interfaces of the a8 and a^ subunits. Once acetylcholine is bound to a receptor, the channel is opened within a few microseconds. Studies measuring the permeability of different small cations suggest that the open ion channel is, at its narrowest, about 0.65-0.80 nm in diameter, in agreement with estimates from electron micrographs. This would be sufficient to allow passage of both Na+ and K+ ions with their shell of bound water molecules. Thus the acetylcholine receptor probably transports hydrated ions, unlike Na+ and K+ channels, both of which allow passage only of nonhydrated ions (see Figure 7-16).

Although the structure of the central ion channel is not known in molecular detail, much evidence indicates that it is lined by five homologous transmembrane M2 a helices, one from each of the five subunits. The M2 helices are

▲ FIGURE 7-45 Three-dimensional structure of the nicotinic acetylcholine receptor. (a) Schematic cutaway model of the pentameric receptor in the membrane; for clarity, the p subunit is not shown. Each subunit contains an M2 a helix (red) that faces the central pore. Aspartate and glutamate side chains at both ends of the M2 helices form two rings of negative charges that help exclude anions from and attract cations to the channel. The gate, which is opened by binding of acetylcholine, lies within the pore. (b) Top: Cross section of the exoplasmic face of the composed largely of hydrophobic or uncharged polar amino acids, but negatively charged aspartate or glutamate residues are located at each end, near the membrane faces, and several serine or threonine residues are near the middle. Mutant acetylcholine receptors in which a single negatively charged glutamate or aspartate in one M2 helix is replaced by a positively charged lysine have been expressed in frog oocytes. Patch-clamping measurements indicate that such altered proteins can function as channels, but the number of ions that pass through during the open state is reduced. The greater the number of glutamate or aspartate residues mutated (in one or multiple M2 helices), the greater the reduction in ion conductivity. These findings suggest that aspartate and glutamate residues form a ring of negative charges on receptor showing the arrangement of subunits around the central pore. The two acetylcholine binding sites are located about 3 nm from the membrane surface. Bottom: Top-down view looking into the synaptic entrance of the channel. The tunnel-like entrance narrows abruptly after a distance of about 6 nm. These models are based on amino acid sequence data, computer-generated averaging of high-resolution electron micrographs, and information from site-specific mutations. [Part (b) from N. Unwin, 1993, Cell 72, and Neuron, 10 (suppl.), p. 31.]

the external surface of the pore that help to screen out anions and attract Na+ or K+ ions as they enter the channel. A similar ring of negative charges lining the cytosolic pore surface also helps select cations for passage (see Figure 7-45).

The two acetylcholine binding sites in the extracellular domain of the receptor lie =4 to 5 nm from the center of the pore. Binding of acetylcholine thus must trigger conforma-tional changes in the receptor subunits that can cause channel opening at some distance from the binding sites. Receptors in isolated postsynaptic membranes can be trapped in the open or closed state by rapid freezing in liquid nitrogen. Images of such preparations suggest that the five M2 helices rotate relative to the vertical axis of the channel during opening and closing (Figure 7-46).

▲ FIGURE 7-46 Schematic models of the pore-lining M2 helices in the closed and opened states. In the closed state, the kink in the center of each M2 helix points inward, constricting the passageway, whose perimeter is indicated by the blue spheres. In the open state, the kinks rotate to one side, so that the helices are farther apart. The green spheres denote the hydroxyl groups of serine (S) and threonine (T) residues in the center of the M2 helices; in the open state these are parallel to the channel axis and allow ions to flow. [Adapted from N. Unwin, 1995, Nature 373:37.]

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