Molecular Properties ofthe Acetylcholineactivated Channel

Techniques of molecular biology are being applied with great success to the study of ion channel function, particularly when combined with measurements of single-channel behavior using the patch-clamp technique just described.

Figure 8-15 The subunit structure of the ACh-activated channel. The five subunits interact to form the gated ion channel of the end-plate membrane, with the pore at the center.

This has been especially true for the ACh-activated channel of the muscle end-plate. Biochemical studies have shown that this channel is formed by the aggregation of five individual protein subunits: two copies of an alpha-subunit, plus beta-, gamma-, and delta-subunits. The two ACh-binding sites have been located, one on each of the two alpha-subunits, thus accounting neatly for the fact that binding of two ACh molecules is required to open the channel (Figure 8-4). The subunits come together as shown in Figure 8-15 to form the ACh-activated channel, with parts of each subunit forming the aqueous pore at the center through which cations can cross the membrane. The genes encoding each of these subunits have been identified and analyzed, and the sequence of amino acids making up the protein has been deduced in each case by reading the genetic code from the pattern of nucleic acids in the DNA. This sequence of amino acids gives valuable structural information about the ACh-activated channel. But beyond that, molecular biological techniques can be used to assign functional roles to particular parts of the channel protein. This approach makes use of the fact that it is possible to make messenger RNA from the DNA sequence of the channel protein; when this mRNA is injected

Figure 8-16 A summary of the sequence of events during synaptic transmission at the neuromuscular junction.

1. Presynaptic action potential

2. Depolarization of synaptic terminal

3. Voltage-sensitive calcium channels open

4. Calcium enters terminal

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