Receptors formed by different combinations of subunits.
Current Concepts and State of Knowledge
Pharmacologic Properties of Neuronal Nicotinic Acetylcholine Receptors
When the French doctor Jean Nicot brought tobacco plant powder to cure the headache of his queen in the sixteenth century, it would have been impossible to predict that Nicot would leave his name in the history of pharmacology. The natural alkaloid in the tobacco plant was termed nicotine; however, it is only centuries later, with the discovery of receptors that are specifically activated by nicotine, nicotinic acetyl-choline receptors (nAChRs), that we are beginning to understand nicotine's effects on the CNS and peripheral nervous system.
nAChRs belong to a superfamiliy of ligand-gated ion channels characterized by a conserved sequence in the N-terminal domain flanked by linked cysteines that also includes 5-HT3, GABA and glycine receptors. These highly conserved, integral membrane proteins are pentameric structures, composed of a and b subunits. To date, nine a (2-7, 9-10) and three b (2, 3, and 4) subunits have been identified in the CNS. nAChRs may consist of five identical subunits (homomeric) or two or more different sub-units (heteromeric). The a7 subunit forms homomeric receptors, a major component of the nAChRs in the CNS. Heteromeric receptors formed by the a4 and b2 subtypes are the principal receptor types in the mammalian brain. Some subunit combinations are more widely expressed in the CNS than others, while some are restricted to well-defined neuronal pathways. For example, the a6-contain-ing receptor, which predominantly results from coassem-bly with a4 and b2 subunits, is preferentially expressed in the mesolimbic system (Albuquerque et al. 2009; Dani and Bertrand 2007). Since receptors can contain multiple a and b subunit types and the subunit composition varies in different species, insight into receptor composition and localization, especially in the human brain, is critically important. However, our knowledge remains limited due to a lack of sufficiently specific antibodies/ligands to detect the receptors.
To understand the relevance of receptor composition for signal transduction during receptor activation, key properties of the ligand-binding site should be examined. nAChR subunits are arranged in a doughnut-like manner, forming an ionic pore in their center. They have an extracellular ligand-binding site, N- and C-termini, and four membrane-spanning domains. Figure 1 is a schematic representation of the receptor. Three loops in the N-terminal of the a subunit form the major component of the ligand-binding site and appose three loops of the adjacent subunit that form the complementary binding site. In this specific arrangement, both subunits interact with the ligand and, therefore, determine the binding properties.
The simplest representation of nAChR ligand binding is a bimolecular reaction, in which the ligand binds and is released with a single association and dissociation constant. Accordingly, ligand affinity is characterized by a binding constant, that is, the equilibrium between the ON and OFF rates. However, such data provide little or no information on the conformational states that are stabilized by ligand binding, so that agonists, which stabilize the open active state, are indistinguishable from antagonists, which
Nicotinic Agonists and Antagonists. Fig. 1. Schematic representation of the nAChR and the ACh binding site. (a) Representation of a single subunit formed of a-helices (cylinders), spanning the membrane four times with the N- and C-termini facing the extracellular domain. Note the position of the second transmembrane domain (TM2) that is facing the ionic pore. (b) Schematic drawing of the nAChR inserted into the cell membrane lipid bilayer represented to scale. The ligand-binding site lies at the interface between two adjacent subunits.
stabilize a closed conformation. Additionally, radioligand binding affinities provide no insight into the functional efficacy needed to classify ligands according to their phar-macologic effects. Typically, ligands are subdivided according to their physiologic effects — full agonists are as efficacious as ► acetylcholine (ACh) in activating the receptor; partial agonists are less efficacious than ACh; and antagonists inhibit ACh receptor activation. Importantly, functional efficacy (the amount of receptor activation at maximal concentration) is not correlated with potency (the concentration of ligand required to cause half activation of the receptor; Fig. 2a). It should be noted that the continuous presence of a partial agonist in the vicinity of the receptor will alter its response to a full agonist (Hogg and Bertrand 2007). Such a condition is typically encountered when a compound is given sys-temically and interacts with the endogenous release of the neurotransmitter.
A further complexity, common to all ligand-gated channels, is that sustained presence of an agonist progressively stabilizes the receptor in a desensitized and nonconducting state. Thus, exposure to an agonist is expected to cause transient activation, followed by sustained inhibition due to desensitization, which typically occurs at concentrations one or two orders of magnitude lesser than those required for activation. This indicates
Nicotinic Agonists and Antagonists. Fig. 2. Concentration activation profiles for nAChR activation and desensitization. (a) Typical concentration activation curves for a full agonist (100% efficacy) and a partial agonist (<100% efficacy) with different EC50 values (dotted arrows). Note that the potency of the compound (EC50) does not correlate with its efficacy (relative activation vs. ACh). (b) Concentration relationships for activation and desensitization are represented on the same graph. Note that desensitization occurs at lower concentrations when compared with that of activation, indicating that sustained exposure to an agonist will mainly cause receptor desensitization.
that ligands display a higher affinity for receptors in the desensitized state than in the active state, which is easily seen when plotting activation and desensitization profiles in one graph (Fig. 2b). Since radioligand binding data are acquired during prolonged incubation (>0.5 h) with the ligand to reach equilibrium, agonist binding affinities correlate best with receptor desensitization potencies.
Transition from the resting (closed) to the active (open) state reflects changes in the three-dimensional structure of the receptor. The probability of conversion from the resting to the active state in the absence of a ligand is very low, as shown by the low frequency and brief duration of channel opening. Though the simplest model involves only one transition between the resting and active states, evidence points toward the existence of an intermediate state,
Nicotinic Agonists and Antagonists. Fig. 3. Activation and modulation of the nAChRs. (a) Schematic representation of the energy barriers between the closed (resting) and the active (open) state. The intermediate state, also referred to as the ''flip state,'' has been recently shown to play an important role for partial agonists. (b) Effects of positive (+) or negative (—) allosteric modulators on the concentration activation profiles. Note that positive allosteric modulators cause an enhancement of the evoked response, the agonist potency, and increase in the apparent cooperativity (slope of the curve).
thought to be closed and termed as the "flip state'' (Fig. 3) (Lape et al. 2008; Mukhtasimova et al. 2009).
The complex receptor structure allows high-affinity binding at sites other than the ligand-binding (► orthosteric) site. Molecules that bind at these so-called ► allosteric sites can either increase (positive ► allosteric modulators) or decrease (negative allosteric modulators) the evoked current and thereby affect nAChR function (Changeux and Edelstein 2005).
nAChR Antagonists and Partial Agonists as Pharmacotherapies
Given the diversity of physiologic roles of specific nAChR subtypes and their relationship with disease states, the
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