The Pentameric Family

The pentameric family of receptors has five membrane-spanning subunits. The best-known example of this type of ion channel receptor is the nicotinic acetylcholine receptor at the neuromuscular junction. It consists of one β, one ε, one δ and two α subunits. Two acetylcholine molecules bind to the α subunits, resulting in a rapid increase in Na⁺ flux through the ion channel formed, leading to membrane depolarisation.

Another familiar member of this family is the GABA_A receptor, in which GABA is the natural ligand. Conformational changes induced when the agonist binds cause a chloride-selective ion channel to form, leading to membrane hyperpolarisation. The benzodiazepines (BDZs) can influence GABA activity at this receptor but augment chloride ion conductance by an allosteric mechanism (see below for explanation).

The 5-HT₃ receptor is also a member of this pentameric family; it is the only serotonin receptor to act through ion-channel opening.

Ionotropic Glutamate

Glutamate is an excitatory neurotransmitter in the central nervous system (CNS) that works through several receptor types, of which N-Methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate are ligand-gated ion channels. The NMDA receptors are comprised of two subunits, one pore-forming (NR1) and one regulatory that binds the co-activator, glycine (NR2). In vivo, it is thought that the receptors dimerise, forming a complex with four subunits. Each NR1 subunit has three membrane-spanning helices, two of which are separated by a re-entrant pore-forming loop. NMDA channels are equally permeable to Na⁺ and K⁺ but have a particularly high permeability to the divalent cation, Ca²⁺. Ketamine, xenon and nitrous oxide are non-competitive antagonists at these receptors.

Ionotropic Purinergic Receptors

This family of receptors includes PX1 and PX2. Each has two membrane-spanning helices and no pore-forming loops. They form cationic channels that are equally permeable to Na⁺ and K⁺ but are also permeable to Ca²⁺. These purinergic receptors are activated by ATP and are involved in mechanosensation and pain. These are not to be confused with the two G-protein coupled receptor forms of purinergic receptors, which are distinguished by selectivity for adenosine or ATP.

G-protein Coupled Receptors and G-proteins

G-protein coupled receptors (GPCRs) are membrane-bound proteins with a serpentine structure consisting of seven helical regions that traverse the membrane. G-proteins are a group of heterotrimeric (three different subunits, α, β and γ) proteins associated with the inner leaflet of the cell membrane that act as universal transducers involved in bringing about an intracellular change from an extracellular stimulus. The GPCR binds a ligand on its extracellular side and the resultant conformational change increases the likelihood of coupling with a particular type of G-protein resulting in activation of intermediate messengers at the expense of guanylyl triphosphate (GTP) breakdown. This type of receptor interaction is sometimes known as metabotropic in contrast with ionotropic for ion-channel forming receptors. As well as transmitting a stimulus across the cell membrane the G-protein system produces signal amplification, whereby a modest stimulus may have a much greater intracellular response. This amplification occurs at two levels: a single activated GPCR can stimulate multiple G-proteins and each G-protein can activate several intermediate messengers.

G-proteins bind GDP and GTP, hence the name ‘G-protein’. In the inactive form GDP is bound to the α subunit but on interaction with an activated GPCR, GTP replaces GDP, giving a complex of α-GTP-βγ. The α-GTP subunit then dissociates from the βγ dimer and activates or inhibits an effector protein, either an enzyme, such as adenylyl cyclase or phospholipase C (Figure 3.2) or an ion channel. For example, β-adrenergic agonists activate adenylyl cyclase and opioid receptor agonists, such as morphine, depress transmission of pain signals via inhibition of N-type Ca²⁺ channels through G-protein mechanisms. In some systems, the βγ dimer can also activate intermediary mechanisms.

Figure 3.2 Effect of ligand-binding to G-protein coupled receptor (GPCR). Ligand binding to the 7-TMD GPCR favours association with the G-Protein, which allows GTP to replace GDP. The α unit then dissociates from the G-protein complex to mediate enzyme and ion-channel activation/inhibition.

The α-subunit itself acts as a GTPase enzyme, splitting the GTP attached to it to regenerate an inactive α-GDP subunit. This then reforms the entire inactive G-protein complex by recombination with another βγ dimer.

The α subunit of the G-proteins shows marked variability, with at least 17 molecular variants arranged into three main classes. G_s type G-proteins have α subunits that activate adenylyl cyclase, G_i have α subunits that inhibit adenylyl cyclase and G_q have α subunits that activate phospholipase C. Each GPCR will act via a specific type of G-protein complex and this determines the outcome from ligand-receptor coupling. It is known that the ratio of G-protein to GPCR is in favour of the G-proteins in the order of about 100 to 1, permitting signal amplification. Regulation of GPCR activity involves phosphorylation at the intracellular carboxyl-terminal that encourages binding of a protein, β-arrestin, which is the signal for removal of the receptor protein from the cell membrane. The binding of an agonist may increase phosphorylation and so regulate its own effect, accounting for tachyphylaxis seen with β-adrenergic agonists.

Adenylyl cyclase catalyses the formation of cAMP, which acts as a final common pathway for a number of extracellular stimuli. All β-adrenergic effects are mediated through G_s and opiate effects through G_i. The cAMP so formed acts by stimulating protein kinase A, which has two regulatory (R) and two catalytic (C) units. cAMP binds to the R unit, revealing the active C unit, which is responsible for the biochemical effect, and it may cause either protein synthesis, gene activation or changes in ionic permeability.

cAMP formed under the regulation of G-proteins is broken down by the action of the phosphodiesterases (PDEs). The PDEs are a family of five isoenzymes, of which PDE III is the most important in heart muscle. PDE inhibitors, such as theophylline and enoximone, prevent the breakdown of cAMP so that intracellular levels rise. Therefore, in the heart, positive inotropy is possible by either increasing cAMP levels (with a β-adrenergic agonist or a non-adrenergic inotrope such as glucagon), or by reducing the breakdown of cAMP (with a PDE III inhibitor such as milrinone).

Phospholipase C is also under the control of G-proteins, but the α subunit is of the G_q type. Activation of G_q-proteins by formation of an active ligand–receptor complex promotes the action of phospholipase C. This breaks down a membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP₂), to form inositol triphosphate (IP₃) and diacylglycerol (DAG).

The two molecules formed have specific actions; IP₃ causes calcium release in the endoplasmic reticulum, and DAG causes activation of protein kinase C, with a variety of biochemical effects specific to the nature of the cell in question. Increased calcium levels act as a trigger to many intracellular events, including enzyme action and hyperpolarisation. Again, the common messenger will cause specific effects according to the nature of the receiving cellular subcomponent.

α₁-Adrenoceptors, the muscarinic cholinergic types 1, 3 and 5 as well as angiotensin II type 1 receptors exert their effects by activation of G_q-proteins.

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