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Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.
Palmer Taylor and Joan Heller Brown .
1 Correspondence to Palmer Taylor and Joan Heller Brown, Department of Pharmacology, 0636, University of California, San Diego, La Jolla, California 92093.
Muscarinic and nicotinic receptors are related more closely to other receptors in their respective families than to one another, both structurally and functionally. The nicotinic receptor is far more similar to other ligand-gated ion channels, such as the GABA receptor, than to the muscarinic receptor. The muscarinic receptor in turn belongs to a group of seven transmembrane-spanning receptors that includes the adrenergic receptors [32], which transduce their signals across membranes by interacting with GTP-binding proteins (see Chap. 20). Several macromolecular interactions are involved in the responses triggered by activation of the muscarinic receptor. These associations contribute to the 100 to 250 msec latency characteristic of muscarinic responses, which are slow compared with those mediated by nicotinic receptors.
Many types of neurons and effector cells respond to muscarinic receptor stimulation. Despite the diversity of responses that ensue, the initial event that follows ligand binding to the muscarinic receptor may be, in all cases, the interaction of the receptor with a G protein. Depending on the nature of the G protein, the receptor—G protein interaction can initiate any of several early biochemical events seen with muscarinic receptor occupation, including inhibition of adenylyl cyclase, stimulation of phosphoinositide hydrolysis or regulation of potassium channels (Fig. 11-9) [33].
Acetylcholine (ACh) interacts with a muscarinic receptor of the subtypes indicated to induce various responses. The M2 and M4 muscarinic acetylcholine receptors (mAChRs) interact with the α subunit of GTP-binding protein, Gi. When ACh binds, Gα (more. )
Decreased cAMP formation is caused by muscarinic receptor stimulation. This effect is most apparent when adenylyl cyclase is stimulated, for example, by activation of adrenergic receptors with catecholamines or forskolin. Simultaneous addition of cholinergic agonists decreases the amount of cAMP formed in response to the catecholamine, in some tissues almost completely. The result is diminished activation of cAMP-dependent protein kinase (PKA) and decreased substrate phosphorylation catalyzed by this kinase. The mechanism by which the muscarinic receptor inhibits adenylyl cyclase is through activation of an inhibitory GTP-binding protein, Gi. The α subunit of Gi competes with the α subunit of the G protein activated by stimulatory agonists (GS) for regulation of adenylyl cyclase (see Chaps. 20 and 22). Although muscarinic receptors do not interact with Gs, increases in cAMP formation are seen under some circumstances. These may result from stimulatory effects of βγ subunits released from Gi or effects of elevated intracellular Ca 2+ on specific isoforms of adenylyl cyclase.
Activation of phosphoinositide-specific phospholipase C by muscarinic agonists stimulates phosphoinositide hydrolysis. Activation of the β1 isoform of phosphoinositide-specific phospholipase C (PI-PLC) is mediated through the α subunit of a GTP-binding protein, Gq/11 [34]. This is the primary mechanism by which muscarinic receptors regulate this enzyme. However, some PLC isoforms, most clearly β2, also are activated by βγ subunits. This probably accounts for the pertussis toxin-sensitive, Gi/Go-mediated activation of PI-PLC seen when high levels of cloned M2 receptors are expressed stably in some cell lines. The hydrolysis of phosphatidylinositol 4,5-bisphosphate yields two potential second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (see Chap. 21). DAG increases the activity of the Ca 2+ and phospholipid-dependent protein kinase (PKC). IP3 mobilizes Ca 2+ from intracellular stores in the endoplasmic reticulum and thereby elevates cytosolic free Ca 2+ . Subsequent responses are triggered by direct effects of Ca 2+ on Ca 2+ -regulated proteins and by phosphorylation mediated through Ca 2+ /calmodulin-dependent kinases and PKC. Stimulation of a phospholipase D, which hydrolyzes phosphatidylcholine, also occurs in response to muscarinic receptor activation. This appears to be secondary to activation of PKC and contributes to a secondary rise in DAG.
Regulation of K + channels. Muscarinic agonists cause rapid activation of G protein-coupled, inwardly rectifying potassium channels (GIRKs). This muscarinic effect can be mimicked by GTP analogs in whole-cell clamp experiments, and the response is sensitive to pertussis toxin, which ribosylates and inactivates Gi and a related protein, Go (see Chap. 20). It is now generally agreed that GIRK1 and GIRK2 are activated directly by binding βγ subunits released from Gi or Go. This is a primary mechanism by which muscarinic agonists cause hyperpolarization of cardiac atrial cells, as well as of neurons [35]. This pathway contrasts with muscarinic inhibition of the M-current in sympathetic ganglia; suppression of this K + channel is mediated indirectly through muscarinic formation of a diffusible second messenger.
Intracellular mediators of muscarinic receptor action. The three events described above, inhibition of adenylyl cyclase, stimulation of PLC and regulation of K + channels, occur within the plasma membrane. They can be triggered directly by muscarinic receptor occupation independent of changes in cytosolic mediators. However, these primary events in turn affect the generation of diffusible second messengers such as cAMP, DAG, IP3 and Ca 2+ , which generate other metabolic sequelae. For example, an increase in cytosolic free Ca 2+ probably contributes to activation of phospholipase A2, generating arachidonic acid, prostaglandins and related eicosanoids (see Chap. 35). These products in turn can stimulate cGMP formation and can regulate ion channel activity. Increased Ca 2+ also can activate Ca 2+ -dependent ion channels (K + , Cl − ), regulate cAMP phosphodiesterase and activate Ca 2+ /calmodulin kinase-dependent protein phosphorylation. PKC is activated by DAG, generally in concert with Ca 2+ , and has effects on ion-channel activity, as well as on cholinergic secretory and contractile responses. Given the obviously complex set of possible interactions between the intracellular mediators, it is easy to explain how diverse cellular responses can be mediated through a single receptor activating relatively few primary responses (see Chap. 10).
In membranes or homogenates from heart, brain and other tissues, muscarinic agonists compete for antagonist-binding sites with Hill slopes of less than unity, suggesting that these agonists interact with more than a single population of muscarinic receptors [32]. Direct binding experiments with radiolabeled agonists also show multiple binding sites for agonists. Competition curves are best fit by a model in which there are sites with low, high and in some cases, superhigh affinity for agonists. Addition of GTP to the binding assay can have a dramatic effect on the agonist competition curve or on direct agonist binding. The effect of GTP is to decrease the apparent affinity of the receptor for agonists. This results from a change in the interaction of the receptor with the GTP-binding protein that transduces its effects.
Agonists vary in their binding properties. Some, like ACh, carbamylcholine and methacholine, bind with high affinity to a large percentage of the total sites. Others, like oxotremorine and pilocarpine, appear to bind to a single class of sites and may show relatively little high-affinity binding. The capacity of an agonist to induce high-affinity binding correlates with the efficacy of that agonist for eliciting responses such as contraction or phosphoinositide breakdown. It therefore appears that interaction of the receptor and G protein is critical to production of the cellular response.
Unlike agonists, most muscarinic antagonists, such as quinuclidinylbenzilate, N-methylscopolamine and atropine, bind to the receptor with Hill slopes of unity, as expected for a mass-action interaction with a single receptor type. There is little difference in affinity for these ligands in various tissues. Similar findings with other antagonists initially suggested that all muscarinic receptors were the same. However, a number of functional studies have suggested that muscarinic receptors are heterogeneous, and several putative subtype-selective antagonists have been described throughout the years.
Pirenzepine (PZ) binds to muscarinic receptors in cortex, hippocampus and ganglia with relatively high affinity; these sites have been termed M1, as mentioned earlier. Heart, gland and smooth muscle muscarinic receptors, as well as those in brainstem, cerebellum and thalamus, show 30- to 50-fold lower affinity for PZ [32,33]. The affinity for classic antagonists like N-methylscopolamine is the same in all of these regions, emphasizing the unique selectivity of PZ. Direct binding studies using [ 3 H]PZ confirm that only certain tissues and brain regions have receptors with high affinity for this antagonist. Results of pharmacological studies also indicate that PZ blocks muscarinic responses in ganglia better than responses in heart. Brain and heart subsequently were used to purify M1 and M2 muscarinic receptors, and cDNA clones corresponding to these receptors were isolated from rat brain and heart libraries.
The cDNAs for the muscarinic receptors encode apparent glycoproteins of 55 to 70 kDa, which contain seven predicted transmembrane-spanning regions, similar to what is seen for the β-adrenergic receptor and other receptors that couple to G proteins (Fig. 11-10). There is only 38% amino acid identity between the proteins cloned from porcine brain and heart. The cDNA encoding the receptor initially cloned from the brain has been termed m1, whereas that cloned from the heart has been termed m2.
Predicted amino acid sequence and transmembrane domain structure of the human M1 muscarinic receptor. Amino acids that are identical among the m1, m2, m3 and m4 receptors are dark orange. The shaded cloud represents the approximate region that determines (more. )
The human and rat homologs of these receptor genes, as well as three additional subtypes termed m3, m4 and m5, subsequently have been cloned and expressed. Comparison of the amino acid sequences of the five muscarinic receptor subtypes suggests that they are members of a highly conserved gene family. The greatest sequence identity is in the transmembrane-spanning regions, whereas the long cytoplasmic loop (i3) between transmembrane domains V and VI varies among the receptor subtypes [32,36]. The cloned receptors, expressed in mammalian cells, show differences in antagonist affinity similar to those of the pharmacologically defined receptors. Thus, the expressed m1 receptor is blocked selectively by PZ, the m2 receptor is blocked by AFDX-116 and methoctramine and the m3 receptor is blocked by hexahydrosiladifenidol [33]. The regions in the receptor responsible for differences in antagonist affinity have not yet been identified clearly. Ligands are believed to bind to the receptor at sites facing the extracellular space but located in a central cavity deep within the bundle formed by transmembrane domains III through VII. The binding site for the covalent antagonist propylbenzilylcholine mustard has been mapped to a particular aspartic acid residue in the third transmembrane region. This amino acid is conserved in all biogenic amine G-protein-coupled receptors. Mutagenesis of this amino acid profoundly affects both agonist and antagonist binding to muscarinic receptors. It is hypothesized that this residue participates in ionic bonding with the ammonium headgroup of the cholinergic ligand [32].
Expression of the cloned receptors in Chinese hamster ovary cells, other mammalian cells and Xenopus oocytes has demonstrated differential coupling of these receptors to cellular responses. In general the m1, m3 and m5 receptors regulate phosphoinositide hydrolysis by stimulating PLC. This occurs through selective coupling of the receptor to a pertussis toxin-insensitive G protein, probably Gq/11, which can activate the β isoform of PLC [34]. Calcium-dependent K + and Cl − channels are activated secondarily to the PLC-mediated increase in intracellular Ca 2+ . In contrast, the m2 and m4 receptors couple through a pertussis toxin-sensitive G protein (Gi) to inhibition of adenylyl cyclase. Regulation of K + channels also is mediated through m2 or m4 receptor interaction with specific pertussis toxin-sensitive G proteins.
Chimeric receptors have been used to determine the regions critical for specifying coupling to particular responses. These studies demonstrate that it is the third intracellular (i3) loop that defines functional specificity [33,36]. A series of amino acids proximal to the transmembrane domain, that is, at the amino- and the carboxy-terminal ends of the i3 loop, carry most of this information. These particular regions are similar in the m1, m3 and m5 receptors and in the m2 and m4 receptors but distinguish these two groups from one another. Both site-directed and random mutagenesis studies have identified specific amino acids at the amino-terminus of the i3 loop which are required for G protein recognition and activation [36,37]. These critical noncharged amino acids are predicted to reside on the hydrophobic face of an α-helical extension of transmembrane domain V. Conserved amino acids in the carboxy-terminus of the i3 loop and adjacent transmembrane domain VI also have been demonstrated to specify coupling to Gi- versus Gq-mediated responses. The hydrophobic regions at the two ends of the i3 loop thus are suggested to form a surface that binds to and discriminates between different classes of G protein [36]. Other regions, including a portion of the second intracellular loop, also contribute to specifying correct G protein coupling.
The selectivity in muscarinic receptor coupling is not absolute. Overexpression of receptors or of particular G proteins supports interactions that may differ from those described above. For example, m2 receptors expressed in Chinese hamster ovary cells not only inhibit adenylyl cyclase but also can stimulate phosphoinositide hydrolysis through a pertussis toxin-sensitive G protein [38]; this is not seen, however, when m2 receptors are expressed in Y1 cells. These findings indicate that caution must be exercised in interpreting data obtained when receptors are expressed, often at high levels, in cells in which they normally do not function.
Muscarinic receptors of the m1, m3 and m5 subclasses induce transformation or cell proliferation, a feature not shared by m2 and m4 receptors [39]. This property has been exploited to develop a high-throughput assay for screening effects of receptor mutations [37]. Mitogenactivated protein kinases (MAP kinases) also are activated by muscarinic receptors, but unlike transformation, this response occurs with receptors of the m2/m4 subtype as well as of the m1/m3 subtype. Notably, induction of cell growth by muscarinic receptor stimulation is cell type-specific and is seen only at high levels of receptor expression [39]. Thus, it is questionable whether there is a physiological role for ACh in growth regulation.
Knowledge of the anatomical distribution and coupling properties of receptor subtypes can indicate which physiological responses they mediate in vivo. However, the lack of good subtype-selective antagonists limits the use of pharmacological approaches to address this question. Generation of transgenic mice in which muscarinic receptors are overexpressed or receptor genes are disrupted by homologous recombination provides a new approach for evaluation of muscarinic receptor function. The m 1 gene has been targeted selectively, and m1 receptor expression in the forebrain was eliminated [40]. Homozygous m1 receptor-deficient mice are completely resistant to seizures produced by pilocarpine, implicating the m1 receptor in this model of epilepsy. Furthermore, inhibition of the M-current in sympathetic ganglia, suggested by previous pharmacological experiments to be m1 receptor-coupled, is ablated in knockout mice. Future development of this approach should provide considerable insight into the distinct roles of the m1, m2 and other muscarinic receptor subtypes in peripheral and central nervous system function.
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