BIOLOGY OF PREJUNCTIONAL AND POSTJUNCTIONAL NICOTINIC RECEPTORS

Immature or Extrajunctional versus Mature or Junctional Isoforms

There are two variants of the postjunctional acetylcholine receptors. The acetylcholine receptor isoform present in the innervated, adult neuromuscular junction is referred to as the adult, mature, or junctional receptor. Another isoform is expressed when there is decreased activity in muscle, as seen in the fetus before innervation; after lower or upper motor neuron injury, burns, or sepsis; or after other events that cause increased muscle protein catabolism.^{[60] [61]} To contrast with the mature or junctional receptors, the other isoform is referred to as the immature, extrajunctional, or fetal form of acetylcholine receptor. Some evidence suggests that the immature isoform is not seen in muscle protein catabolism and wasting occurring with malnutrition.^[61] The differences in the protein structure of the two isoforms cause significant qualitative variations among the responses of individual patients to relaxants and seem to be responsible for some of the anomalous results that are observed when administering relaxants to particular individuals. These qualitative differences in the isoforms can also cause variations in function of muscle (see "Myopathy of Critical Illness and Acetylcholine Receptors").^[37]

In addition to their structural compositions, the two isoforms have other characteristics that are different.^{[1] [8] [60]} At the molecular level, both types of receptors consists of five subunits (see Fig. 22-4 ). The mature junctional receptor is a pentamer of two α-subunits and one each of the β-, δ-, and epsilon-subunits. The immature receptor consists of two α-subunits and one each of β-, δ-, and γ-subunits; that is, in the immature receptor, the γ-subunit is present instead of the epsilon-subunit. The γ- and epsilon-subunits differ from each other very little in amino acid homology, but the differences are great enough to affect the physiology and pharmacology of the receptor and its ion channel. Although the names junctional and extrajunctional imply that each is located in the junctional and extrajunctional areas, this is not strictly correct. Junctional receptors are always confined to the end plate (perijunctional) region of the muscle membrane. The immature, or extrajunctional, receptor may be expressed anywhere in the muscle membrane. Despite the name extrajunctional, they are not excluded from the end plate. During development and in certain pathologic states, the junctional and extrajunctional receptors can coexist in the perijunctional area of the muscle membrane ( Fig. 22-8 ).

Quite unlike other cells, muscle cells are unusual in that they have many, usually hundreds of, nuclei per cell. Each of these nuclei has the genes to make both types of receptors. Multiple factors, including electrical activity, growth factor signaling (e.g., insulin, agrin, ARIA), and the presence or absence of innervation, control the expression of the two types of receptor isoforms.^{[8] [10] [25] [46]} This is most clearly seen in the developing embryo as the neuromuscular junction is formed. Before they are innervated, the muscle cells of a fetus synthesize only the immature receptors—hence the term fetal isoform of receptor. The synthesis is directed by nearly all the nuclei in the cell, and the receptors are expressed throughout the membrane of the muscle cell (see Fig. 22-8 ). As the fetus develops and the muscles become innervated, muscle cells begin to synthesize the mature isoform of receptors, which are inserted exclusively into the developing (future) end plate area. The nerve releases several growth factors that influence the synthetic apparatus of the nearby nuclei. First, nerve-supplied factors induce the subsynaptic nuclei to increase synthesis of the acetylcholine receptors. Next, the nerve-induced electrical activity results in repression of receptors in the extrajunctional area. The nerve-derived growth factors, including agrin and ARIA/neuregulin, cause the receptors to cluster in the subsynaptic area and prompt expression of the mature isoform^{[8] [46]} (see Fig. 22-5 ). In conditions associated with insulin resistance, there seems to be proliferation of acetylcholine receptors beyond the junctional area. Conditions in which insulin resistance (i.e., decreased growth factor signaling) has been observed include immobilization, burns, and denervation.^{[62] [63] [64]} In these conditions, there is associated upregulation of the acetylcholine receptors and expression of the immature isoforms.^{[61] [65] [66]}

Before innervation, acetylcholine receptors are present throughout the muscle membrane. After innervation, the acetylcholine receptors become more and more concentrated at the postsynaptic membrane and are virtually absent in the extrasynaptic area at birth. The innervation process progresses somewhat slowly during fetal life and matures during infancy and early childhood.^{[8] [20] [25]} With time, the immature receptors diminish in concentration and disappear from the peripheral part of the muscle. In the active, adult, normal, innervated muscle, only the nuclei under and very near the end plate direct the synthesis of receptor; only the genes for expressing the mature receptors are active. The nuclei beyond the junctional area are not active, and therefore no receptors are expressed anywhere in the muscle cells beyond the

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Figure 22-8 Distribution of acetylcholine receptors in developing adult, mature, and denervated muscle. A and B, In the early fetal stage, mononucleated myoblasts, derived from the mesoderm, fuse to form multinucleated myotubes. The γ-subunit-containing immature acetylcholine receptors are scattered throughout the muscle membrane. C, As the nerve makes contact with muscle, clustering of the receptors occurs at the synapse and is associated with some loss of extrasynaptic receptors. D, Maturation of the junction is said to occur when epsilon-subunit-containing receptors replace the γ-subunit-containing receptors. Even mature muscle is multinucleated, but it is devoid of extrasynaptic nuclei. E, Denervation or another pathologic state (e.g., burns, immobilization, chronic muscle relaxant therapy, sepsis) leads to re-expression of the γ-subunit receptor at the junctional and the extrajunctional areas. The latter changes are potentially reversible.

Proteins implicated in the linking of the mature receptors to the cytoskeleton include utrophin, α- and β-dystroglycan, and rapsyn. Several lines of evidence indicate that the clustering, expression, and stabilization of the mature receptors are triggered by at least three growth factors: agrin, ARIA, and calcitonin gene-related peptide.^{[8] [11]} Agrin is also released from the muscle, but muscle-derived agrin does not seem to be as important in the clustering and maturation of the receptor. ARIA is made in the nerve and seems to play a role in the maturation of vesicular arrangement and conversion of the γ to epsilon switch.^{[46] [67]} All of these growth factors interact with distinct membrane and cytosolic receptor proteins, causing phosphorylation, activation of nuclear (gene) transcriptional systems. Agrin signals through MuSK and ARIA through ERBB receptors (see Fig. 22-5 ). These receptors control qualitative and quantitative changes at the junction. Once begun, the process is very stable, and the nuclei in the junctional area continue to express mature receptors.

The extrajunctional receptors can reappear soon after upper and lower motor denervation and in certain pathologic states (e.g., burns, immobilization, chronic muscle relaxant therapy, loss of electrical activity). Stimulating a denervated muscle with an external electrical stimulus can prevent the appearance of the immature receptors. It has been suggested that the calcium that enters the muscle during activity is important to the suppression process.^[68] In the pathologic states previously enumerated, if the process is severe and prolonged, extrajunctional receptors are inserted all over the surface of the muscle, including the perijunctional area (see Fig. 22-8 ). The junctional nuclei also continue to make mature receptors. The end plates consist of mature and immature receptors. The synthesis of immature receptors is initiated within hours of inactivity, but it takes several days for the whole muscle membrane to be fully covered with receptors. This upregulation of receptors has implications for the use of depolarizing and nondepolarizing relaxants.

The changes in subunit composition (γ versus epsilon) in the receptor confer certain changes in electrophysiologic (functional), pharmacologic, and metabolic characteristics.^{[1] [25]} The mature receptors are metabolically stable, with half-life approximating 2 weeks, whereas the immature receptor has a metabolic half-life of less than 24 hours. Immature receptors have a smaller single-channel conductance and a 2- to 10-fold longer mean channel open time than mature receptors (see Fig. 22-4 ). The changes in subunit composition may also alter the sensitivity or affinity, or both, of the receptor for specific ligands. Depolarizing or agonist drugs such as succinylcholine and acetylcholine depolarize immature receptors more easily, resulting in cation fluxes; one-tenth to one-hundredth

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The sensitivity to muscle relaxants may occur in only certain parts of the body or certain muscles if only some muscles are affected by the diminution of nerve activity (e.g., after a stroke). The sensitivity to relaxants can begin to change between 24 and 72 hours after an injury or hospitalization. The most serious side effect with the use of succinylcholine in the presence of upregulated receptors in one or more muscle is hyperkalemia.^{[1] [2] [69]} In these subjects, the receptors can be scattered over a large surface of the muscle. Immature receptors are especially sensitive to succinylcholine. The channels opened by the agonist allow potassium to escape from the muscle and enter the blood. If a large part of the muscle surface consists of upregulated (immature) receptor channels, each of which stays open for a longer time, the amount of potassium that moves from muscle to blood can be very large. The resulting hyperkalemia can cause dangerous disturbances in cardiac rhythm, including ventricular fibrillation. Moreover, it is difficult to prevent the hyperkalemia by the prior administration of nondepolarizers because extrajunctional receptors are not very sensitive to block by nondepolarizing relaxants.^[1] Larger than normal doses of nondepolarizers may attenuate the increase in blood potassium but cannot completely prevent it. However, hyperkalemia and cardiac arrest can occur after succinylcholine administration, even in the absence of denervation states. This is seen in certain congenital muscle dystrophies, in which the muscle membrane is prone to damage by succinylcholine releasing potassium into the circulation. ^[69]