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Section I:
Cellular and Molecular Neurobiology

4. Synaptic Transmission at the Skeletal Neuromuscular Junction
Part 2 of 2

Overview of the Sequence of Events Underlying the EPP

Figure 4.5

What are the other steps in the process of chemical synaptic transmission? Figure 4.5 provides an overview. A nerve action potential that is initiated in the cell body of a spinal motor neuron propagates out the ventral roots and eventually invades the synaptic terminals of the motor neurons. As a result of the action potential, the chemical transmitter acetylcholine (ACh) is released into the synaptic cleft. ACh diffuses across the synaptic cleft and binds to special receptors on the postsynaptic or the postjunctional membrane. The binding of ACh to its receptors produces a conformational change in a membrane channel that is specifically permeable to both Na⁺ and K⁺. As a result of an increase in Na⁺ and K⁺ permeability, there is a depolarization of the postsynaptic membrane. That depolarization is called the endplate potential or more generally the EPSP. If the EPSP is sufficiently large, as it normally is at the neuromuscular junction, it leads to initiation of an action potential in the muscle cell. The action potential initiates the process of excitation contraction coupling and the development of tension. The duration of the endplate potential is about 10 msec.

Two factors control the duration of the EPSP at the neuromuscular junction. First, ACh is removed by diffusion. Second, a substance in the synaptic cleft, called acetylcholinesterase (AChE), hydrolyzes or breaks down ACh.

Role of AChE

Figure 4.6

An important family of substances, one of which is neostigmine, inhibits the action of AChE. Neostigmine blocks the action of AChE, and thereby makes the endplate potential larger and longer in duration. This figure illustrates two endplate potentials. One was recorded in saline and curare and a second recorded after neostigmine was added to the solution. (Curare is added so that the properties of the EPP can be studied without triggering an action potential in the muscle cell.) After applying neostigmine the endplate potential is much larger and longer in duration.

Myasthenia Gravis

Myasthenia gravis is associated with severe muscular weakness because of a decrease in the number of acetylcholine receptors in the muscle cell. If the endplate potential is smaller, the endplate potential will fail to reach threshold. If it fails to reach threshold, there will be no action potential in the muscle cell and no contraction of the muscle, which causes muscular weakness. Neostigmine and other inhibitors of AChE are used to treat patients with myasthenia gravis. These agents make the amount of acetylcholine that is released more effectively reach the remaining acetylcholine receptors.

Iontophoresis of ACh

Iontophoresis is an interesting technique that can be used to further test the hypothesis that ACh is the neurotransmitter substance at the neuromuscular junction. If ACh is the transmitter that is released by this synapse, one would predict that it should be possible to substitute artificial application of the transmitter for the normal release of the transmitter. Since ACh is a positively charged molecule, it can be forced out of a microelectrode to simulate the release of ACh from a presynaptic terminal.

Figure 4.7

Figure 4.8

Indeed, minute amounts of ACh can be applied to the vicinity of the neuromuscular junction. This figure compares an EPP produced by stimulation of the motor axon and the response to ejections of ACh. The potential change looks nearly identical to the endplate potential produced by the normal release of ACh. This experiment provides experimental support for the concept that ACh is the natural transmitter at this synapse.

The response to the ejection of ACh has some other interesting properties that are all consistent with the cholinergic nature of the synapse at the skeletal neuromuscular junction. Neostigmine makes the response to the iontophoresis of ACh longer and larger. Curare reduces the response because it competes with the normal binding of ACh. If ACh is ejected into the muscle cell, nothing happens because the receptors for acetylcholine are not in the inside; they are on the outside of the muscle cell. Application of acetylcholine to regions of the muscle away from the end-plate produces no response because the receptors for the ACh are concentrated at the synaptic region.

To test your understanding so far, consider how an agent such as TTX would affect the generation of both an EPP and the response of a muscle fiber to the iontophoretic application of ACh? TTX has no effect on the response to ACh, but it does block the EPP. The reason the response to ACh is unaffected is clear, but many expect that if there is no effect here, there should be no effect on the EPP either. Tetrodotoxin does not affect the binding of acetylcholine to the receptors and therefore will not affect the response to direct application of ACh. However, tetrodotoxin will affect the ability of an action potential to be elicited in the motor axon. If an action potential cannot be elicited in the motor axon, it cannot cause the release of transmitter. Thus, tetrodotoxin would totally abolish the EPP. The block would not be due to a block of ACh receptors, but rather to a block of some step prior to the release of the transmitter.

Ionic Mechanisms of the EPP

Bernard Katz and his colleagues were pioneers in investigating mechanisms of synaptic transmission at the neuromuscular junction. They suggested that the channel opened by ACh was one that had equal permeability to both Na⁺ and K⁺. Because it was equally permeable to Na⁺ and K⁺, Katz suggested that, as a result of the opening of these channels, the membrane potential would move toward 0 mV. (A value of alpha in the GHK equation equal to one, which when substituted into the equation, yields a potential of about 0 mV.)

Figure 4.9

The experiment shown in the figure on the left tests that concept. The muscle cell has been penetrated with a recording electrode as well as another electrode that can be connected to a suitable source of potential in order to artificially change the membrane potential. Normally, the membrane potential is about -80 mV [Skeletal muscle cells have higher (more negative) resting potentials than most nerve cells.] Again, a small amount of curare is added so that the EPP is small. Katz noticed in these experiments that the size of the EPP changed dramatically depending upon the potential of the muscle cell. If the membrane potential is moved to 0 mV, no potential change is recorded whatsoever. If the membrane potential is made +30 mV, the EPP is inverted. So three different stimuli produce endplate potentials that are very different from each other.

The lack of a response when the potential is at 0 mV is particularly informative. Consider why no potential change is recorded. Presumably, the transmitter is being released and binding to the receptors. The simple explanation for a lack of potential change is that the potential at which the opening of ACh channels are trying to reach has already been achieved. If the membrane potential is made more positive than 0 mV, then the EPP is inverted. No matter what the potential, the change in permeability tends to move the membrane potential towards 0 mV! If the resting potential is more negative than 0 mV, there is an upward deflection. If it is more positive, there is a downward deflection. If it is already at 0 mV, there is no deflection.

This potential is also called the reversal potential, because it is the potential at which the sign of the synaptic potential reverses. The experiment indicates that, as a result of ACh binding to receptors, specific channels become equally permeable to Na⁺ and K⁺. This permeability change tends to move the membrane potential from wherever it is initially towards a new potential of 0 mV.

Why does the normal endplate potential never actually reach 0 mV? One reason is that the sequence of permeability changes that underlie the action potential "swamp out" the changes produced by the EPP. But even if an action potential was not triggered, the EPP still would not reach 0 mV. This is because the ACh channels are only a small fraction of the total number of channels in muscle fibers. The K⁺ channels that endow the muscle cells with its resting potential are present as well. Their job is to try to maintain the cell at the resting potential.

Figure 4.10

The channel opened by ACh is a member of a general class of channels called ligand-gated channels or ionotropic receptors. As illustrated in Figure 4.10, the transmitter binding site is part of the channel itself. As a result of transmitter binding to the receptor (generally two molecules are necessary), there is a conformational change in the protein allowing a pore region to open and ions to flow down their electrochemical gradients. Additional details of the channel are presented in Chapter 11, Part 5.

Test Your Knowledge

4. An endplate potential in a skeletal muscle cell could in principle be produced by a decreased permeability to which of the following ions(s)? (Assume that there is a finite initial permeability to each of the ions listed below and that physiological concentration gradients are present.):

⁺

²⁺

⁺

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