TRANSMISSION AT THE NEUROMUSCULAR JUNCTION A self-instructional package

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1 TRANSMISSION AT THE NEUROMUSCULAR JUNCTION A self-instructional package Howard Kutchai Department of Molecular Physiology & Biological Physics University of Virginia copyright Howard Kutchai, 2003

2 INTRODUCTION A neuromuscular junction is a synapse between a motoneuron and a muscle cell it innvervates. The prejunctional nerve terminal is a transducer that converts an electrical signal (an action potential in the prejunctional motoneuron) into a chemical signal (transmitter release). The postjunctional muscle cell responds to the transmitter by depolarizing and this results in an action potential in the muscle cell membrane, which triggers muscle contraction. The neuromuscular junction is also important as a model synapse. Because neuromuscular junction is more convenient for experimentation that central vertebrate synapse between neurons, much of what is known of chemical synapses in general was first learned by studying the neuromuscular junction. LEARNING OBJECTIVES 1. List the major events that occur in transmission at a chemical synapse. 2. List the specific events that occur at the neuromuscular junction (motor endplate). 3. State that acetylcholine is released in discrete packets called "quanta", each quantum corresponding to one presynaptic vesicle. 4. Define the miniature endplate potential and state that is due to the spontaneous release of one quantum (vesicle) of acetylcholine. 5. State that at the post-synaptic membrane acetylcholine causes the membrane to become equally permeable to Na + and K + so that the reversal potential of the endplate potential is the average of the equilibrium potentials for Na + and K +. Given E Na and E K use the chord conductance equation to compute the reversal potential of the endplate potential. 6. State that transmitter action is terminated by ACh hydrolysis by acetylcholine-esterase that is located on the postsynaptic membrane. Know that anti-cholinesterases enhance and prolong the endplate potential. 7. State that much of the choline liberated in the synaptic cleft is actively taken back up by the presynaptic terminal to be used in resynthesizing ACh. State that acetylcholine in synthesized from acetyl CoA and choline by the enzyme choline-o-acetyltransferase in the prejunctional nerve terminal. 1

3 PRACTICE CYCLE 1 INPUT 1 A synapse is a junction between excitable cells that is specialized for communication between the cells. There are two types of synapse: electrical and chemical synapses. At an electrical synapse electric current from one cell passes electrotonically to another. Such synapses are also called ephaptic. Electrical synapses have junctional structures like gap junctions (figure to the right) that have a low electrical resistance. The gap junction channels pass molecules with MW as large as 1500 and thus have a very low electrical resistance. Gap junction channels open and close in response to changes in ph, [Ca 2+ ], and other signals. The figure shows a model for the opening and closing of the channels. At a chemical synapse there is some sort of enlargement of the presynaptic axon which contains vesicles filled with the transmitter substance. The presynaptic cell is separated by the synaptic cleft from the postsynaptic cell. The membrane of the postsynaptic cell is also structurally specialized at the synapse. The chemical synapses we know of all have certain features in common. The following sequence of events occurs at chemical synapses: 1. Action potential in presynaptic cell. 2. Depolarization of plasma membrane in presynaptic axon terminal. 3. Release of transmitter substance by presynaptic nerve terminals. 4. Diffusion of transmitter across synaptic cleft. 5. Chemical combination of transmitter with receptor molecule on postsynaptic membrane. 6. Transient change in conductance of postsynaptic membrane to one or more ions. 7. Transient change in the membrane potential of the postsynaptic cell. 2

4 PRACTICE 1 List (without looking at the list provided) the sequence of events that occurs during transmission at a chemical synapse. FEEDBACK 1 The properties you listed apply to chemical synapses in general. The vertebrate chemical synapse about which we have the most direct information is the synapse between a motor nerve terminal and the striated muscle cell it innervates. This synapse is called the neuromuscular junction, the myoneural junction, or the motor endplate. Much that we know about vertebrate synapses was first learned by studying the neuromuscular junction. PRACTICE CYCLE #2 INPUT 2 The terminal branches of a motor axon lose their myelin sheaths. At the neuromuscular junction (figure to the right) the terminal branch of the motor axon lies in a shallow groove, or synaptic trough, on the muscle cell. The muscle plasma membrane that forms the synaptic trough is pleated to form postjunctional folds. The transmitter at the neuromuscular junction is acetylcholine, which is contained in vesicles in the prejunctional nerve terminals. Acetylcholine receptor proteins are concentrated on the crests of the postjunctional folds (inset). The sequence of events that occurs at the neuromuscular junction when an action potential arrives in the presynaptic nerve terminal is as follows: 1. Action potential in the motor axon is conducted into presynaptic nerve terminal. 2. Increase in Ca 2+ conductance of the prejunctional membrane and an inrush of Ca 2+ into the nerve terminal. 3. Fusion of presynaptic vesicles with plasma membrane of motor nerve terminal and release of acetylcholine by exocytosis. 4. Diffusion of acetylcholine across junctional cleft. 5. Binding acetylcholine to specific receptor protein molecules on postjunctional membrane. 3

5 6. Increase in conductance of postjunctional membrane to both Na + and K Transient depolarization of the postjunctional membrane known as the endplate potential. 8. Electrotonic depolarization of areas of the adjacent muscle cell plasma membrane until threshold is reached. 9. Propagation of an action potential by the muscle cell membrane that travels away from the motor endplate in both directions. 10. Termination of the action of acetylcholine by its hydrolysis by acetylcholinesterase. PRACTICE 2 List the sequence of events that occurs at the neuromuscular junction when an action potential arrives at the prejunctional nerve terminal. FEEDBACK 2 The endplate potential depolarizes the postjuctional membrane by about 20 mv. This is sufficient to bring the plasma membrane that is adjacent to the neuromuscular junction threshold so that action potentials are generated. The neuromuscular junction is typically near the middle of the long skeletal muscle cells. Action potential are conducted rapidly towards ends of the muscle cell and toward the center of the muscle cell via the T-tubules. This permits the almost simultaneous contraction of all the sarcomeres of the muscle cell. PRACTICE CYCLE #3 INPUT 3 If exocytosis is the only mechanism by which acetylcholine is released from motor nerve terminals, then the smallest number of acetylcholine molecules that can be released is the number in one presynaptic vesicle. Thus we speak of transmitter released being quantized, with one quantum being the contents of one synaptic vesicle. The quantal nature of transmitter release has functional consequences. Even when no action potential occurs in the presynaptic motor axon, small depolarizations are observed to occur in the postsynaptic muscle cell. 4

6 Even when no action potential occurs in the presynaptic motor axon, small depolarizations are observed to occur in the postsynaptic muscle cell. These depolarizations are much too small (about 0.4 mv) to cause an action potential in the muscle cell and they are called miniature endplate potentials (MEPPs). MEPPS occur randomly with an average frequency of about 1 per second. The MEPP has the same time course as the endplate potential itself and is altered in similar ways by pharmacologic agents that alter the endplate potential. It appears that each MEPP results from the spontaneous release of one quantum of acetylcholine consequences. PRACTICE 3 (a) What is a quantum? (b) What is a miniature endplate potential? (c) How/why does a miniature endplate potential occur? FEEDBACK 3 A quantum is the amount of acetylcholine in one prejunctional vesicle. A MEPP is the small postjunctional depolarization caused by the spontaneous release from the prejunctional terminal of one quantum. The function of the miniature endplate potential remains unknown, but it has often been speculated that MEPPs play a role in the communication between nerve and muscle that is responsible for the trophic influence of nerve or muscle. The MEPP a natural phenomenon that has given us insight into the nature of synaptic transmission. PRACTICE CYCLE #4 INPUT 4 In general the amount of acetylcholine released in response to a single action potential in the prejunctional axon is fairly constant, so that the resulting endplate potential is relatively constant in size. However, the pattern of presynaptic inputs can influence the amount of transmitter released. If a motor axon is stimulated by a series of stimuli, the endplate potential evoked by the stimuli may increase in amplitude with successive stimuli, a phenomenon called facilitation. Facilitation 5

7 is illustrated by the endplate potentials in the figure below. Note that the degree of facilitation depends on the frequency of presynaptic action potentials. After a long period of high frequency stimulation the amplitude of the endplate potential will eventually decline. This is known as neuromuscular depression. If the motor nerve is stimulated tetanically, i.e. stimulated by a large number of stimuli in rapid succession, and then allowed a brief rest, the endplate potential evoked by a single action potential after the period of tetanic stimulation will be larger than that evoked by a single stimulus before the tetanic stimulation. This phenomenon is called post-tetanic potentiation. The figure to the right shows single endplate potentials before and after titanic stimulaton. Apparently, more transmitter is released by a single action potential after tetanic stimulation. The mechanism for this effect remains incompletely understood. Note that the enhanced endplate potential amplitude is fairly long-lived. Facilitation, by contrast, persists for less than 1 sec after repetitive stimulation ceases. 6

8 PRACTICE 4 (a) What is facilitation? (b) What is neuromuscular depression? (c) Define post-tetanic potentiation FEEDBACK 4 Did you say something like the following? a) Facilitation is the phenomenon whereby repetitive single presynaptic impulses lead to progressivley increased amplitudes of the endplate potentials evoked. b) Neuromuscular depression refers to decreased ampitudes of endplate potentials after stimulation of the motor neuron for a long time at relatively high frequency c) After tetanic stimulation of the motor neuron, a single action potential in the motor neuron will evoke a larger endplate potential than it did before tetanic stimulation. This is called post-tetanic potentiation. PRACTICE CYCLE #5 INPUT 5 We mentioned earlier that binding of acetylcholine to the acetylcholine receptor proteins in the postjunctional membrane results in the increased conductance of the postjunctional membrane to both Na + and K +. During the endplate potential Na + is rushing in through the postjunctional membrane tending to depolarize the cell and K + is rushing out tending to repolarize the cell. The membrane potential attained during the endplate potential is a resultant of these two tendencies. Since Na + and K + are dominating the situation the membrane potential sought by the postjunctional cell can be described by the short form of the chord conductance equation: E m = g K gk + g Na E K gna + g + g K Na E Na If the conductance to Na + and K + are equal, the membrane potential sought will be the average of the equilibrium potentials of Na + and K +. This potential that is 'sought' by the postjunctional cell is sometimes called the reversal potential of the neuromuscular junction. When E m = E rev, the inward flux of Na + tending to depolarize the cell is exactly equal to the outward flux of K + tending to repolarize the cell, so that the net membrane current is zero. Note that if Na + were the only ion involved in the endplate potential, then the reversal potential would be about +65 mv (the Na + equilibrium potential). 7

9 PRACTICE 5 (a) State in words the relationship between the membrane potential sought during the endplate potential and the equilibrium potentials for Na + and K +. (b) If for the postjunctional membrane E Na = +65 and E K = -100, what is the reversal potential of the endplatepotential? FEEDBACK 5 (a) Did you say that the potential sought is the average of the equilibrium potentials of Na+ and K+? Good. This is because the conductances to these two ions across the postjunctional membrane are about equal and dominate the current flow during the endplate potential. (b) Did you say Em = gk EK + g Na E Na = EK + E Na = = 17.5mV g g g g 2 K + Na K + Na Since at other synapses (besides the neuromuscular junction) other ionic conductances are involved, other synapses have different reversal potentials. Knowing the reversal potential may help to characterize the particular synapse and to give important clues as to which ion(s) is (are) involved in the postsynaptic potential response. If only one ion predominates in the postsynaptic response, the reversal potential will be equal to the equilibrium potential for that ion PRACTICE CYCLE 6 INPUT 6 The postsynaptic membrane has a high concentration of acetylcholinesterase molecules bound to the surface of the membrane facing the synaptic cleft. This enzyme hydrolyzes acetylcholine to choline and acetate and destroys its transmitter action. Acetylcholinesterase plays an important role in terminating the action of acetylcholine and making the endplate potential a brief event. PRACTICE 6 (a) What is responsible for the time course of the endplate potential (in a qualitative sense)? (c) How would the endplate potential be altered by the presence in the synaptic cleft of an anticholinesterase (an inhibitor of acetylcholinesterase)? FEEDBACK 6 (a) The time course of the endplate potential reflects the amount of acetylcholine released (and the kinetics of its release) and the rate at which acetylcholinesterase destroys the transmitter. The number of quanta of acetylcholine released due to a single action potential in the prejunctional nerve terminal is about 50 (corresponding to hundreds of thousands of molecules of acetylcholine). There is a delay, the so-called synaptic delay, of about 0.5 msec between the time of depolarization of the prejunctional terminals and the onset of postjunctional depolarization. This delay is believed 8

10 to be due primarily to the time between prejunctional depolarization and transmitter release. The rate of hydrolysis of acetylcholine by the cholinesterase is a prime determinant of the kinetics of the repolarization phase of the endplate potential. (b) An anticholinesterase will increase both the magnitude and duration of the endplate potential, since acetylcholine will reach a higher peak concentration in the cleft and acetylcholine will persist longer when the enzyme inhibitor is present. Anticholinesterases are useful clinically in the diagnosis and treatment of myasthenia gravis. Myasthenic patients have diminished endplate potentials primarily because they possess fewer postjunctional acetylcholine receptor molecules. In the presence of an anticholinesterase the motor endplate of a myasthenic patient function more normally. PRACTICE CYCLE #7 FEEDBACK 6 Acetylcholine is synthesized in the prejunctional nerve terminals from acetyl CoA and choline. The enzyme choline-o-acetyltransferase catalyzes the transfer of the acetyl moiety from acetyl CoA to choline and this enzyme is present in the prejunctional nerve terminals. Acetyl CoA is produced in the prejunctional terminal as a metabolic intermediate. The prejunctional cell cannot, however, synthesize choline which it must obtain from other cells that do synthesize it. In order to conserve choline, the prejunctional plasma membrane has a Na + -powered secondary active re-uptake system which takes back into the cell about half of the choline liberated in the synaptic cleft by acetylcholine hydrolysis. PRACTICE 7 (a) What are the immediate precursors of acetylcholine? Which are products of the metabolism of the pre-junctional nerve terminal? (b) What is the name of the enzyme that catalyzes the syntesis of acetylcholine? (c) What is the fate of choline liberated in the synaptic cleft? 9

11 FEEDBACK 7 (a) choline and acetyl-coa are the precursors (b) choline-o-acetyltransferase is the enzyme (c) about half the choline liberated is taken back up into the presynaptic terminal by an active transport mechanism. Hemicholiniums are drugs that block the reuptake of choline by the prejunctional cell. Treatment with hemicholiniums will ultimately lead to a decrease in the transmitter content of individual quanta since sufficient choline for the synthesis of normal levels of acetylcholine will not be available. The decrease in the amount of acetylcholine in prejuctional vesicles will not occur until the pool of normal presynaptic vesicles that existed at the time the hemicholinium was added has been exhausted. 10

12 POST-TEST 1. List the general sequence of events that occur at any chemical synapse. 2. List the events that occur in chemical transmission at the neuromuscular junction. 3. (a) Define a quantum in terms of the neuromuscular junction. (b) Define the miniature endplate potential (MEPP). (c) What are MEPPs due to? 4. (a) Define facilitation. (b) Define post-tetanic potentiation. 5. (a) What is the relative permeability of the postjunctional membrane to Na + and K + during the endplate potential? (b) How does the reversal potential (or reversal potential) of the endplate potential relate to the equilibrium potentials for Na + and K +? (c) If E Na is +60 mv and E K is -90 mv, what is the approximate reversal potential for the endplate potential? 6. (a) What terminates the endplate potential? (b) What effect will an anticholinesterase have on the endplate potential? 7. (a) What is the fate of choline that is formed by the action of the cholinesterase present on the postsynaptic membrane? (b) What are the immediate precursors of acetylcholine? (d) What is the name of the final enzyme involved in acetylcholine synthesis? Where is this enzyme present? 11

13 ANSWERS TO POST-TEST 1. Action potential in presynaptic cell. Depolarization of plasma membrane in presynaptic axon terminal. Diffusion of transmitter across synaptic cleft. Chemical combination of transmitter with receptor molecules on postsynaptic membrane. Transient change in conductance of postsynaptic membrane. Transient change in potential of post-synaptic membrane. 2. Increase in Ca 2+ conductance of prejunctional membrane and increased [Ca 2+ ] in nerve terminal. Fusion of prejunctional vesicles with plasma membrane of motor nerve terminal and release of acetylcholine by exocytosis. Diffusion of acetylcholine across junctional cleft. Binding of acetylcholine to specific receptor protein molecules on postjunctional membrane. Increase in conductance of postjunctional membrane to both Na + and K +. Transient depolarization of the postjunctional membrane known as the endplate potential. Electrotonic depolarization of adjacent areas of the muscle cell plasma membrane until threshold is reached. Propagation of an action potential in the muscle cell membrane that travels away from the motor endplate in both directions. 3. a) A quantum is the smallest amount of acetylcholine that can be released by the presynaptic nerve terminal, the contents of a single presynaptic vesicle. b) A MEPP is a spontaneously occurring small depolarization of the postsynaptic muscle cell at the motor endplate. c) MEPPs are due to spontaneous release of quanta of acetylcholine. 12

14 4. a) In facilitation repetitive stimulation of the motor nerves may lead to endplate potentials that increase in size with each stimulus. b) When a motor nerve is stimulated tetanically (stimulation with an high frequency volley c) of stimuli), the endplate potential evoked by a single stimulus in the motor nerve may be larger following the tetanic stimulation than before. This is called post-tetanic potentiation. 5. a) During the endplate potential the postsynaptic membrane has approximately equal permeability to Na + and K +. b) The reversal potential is close to mid-way between (the average) of the equilibrium potentials for Na+ and K+. c) The reversal potential is about ( )/2 = -15mV. 6. a) The hydrolysis of acetylcholine by cholinesterase terminates the endplate potential. b) An anticholinesterase will increase the magnitude and duration of the endplate potential. 7. a) About half the choline released by hydrolysis is taken back up into the prejunctional nerve terminal by a secondary active transport process. b) Choline and acetyl CoA. c) Choline -o- acetyltransferance. It is found in the motor nerve terminal. 13