Ionotropic and metabotropic receptors LESSON NR PSYCHOBIOLOGY

Transcription

1 Ionotropic and metabotropic receptors LESSON NR PSYCHOBIOLOGY

2 Channels regulated by ligand IONOTROPIC RECEPTORS They are membrane protein complexes, characterized by the presence on their surface not only of the aqueous pore, but also of a specific region, said receptor site (binding site), corresponding to a sort of pocket that receives in a stereo-specific manner one molecule, generally called ligand. Are opposed to the metabotropic receptors that instead indicate a category of receptors, aqueous pore-free and acting through a cascade of events, on ionotropic receptors.

3 IONOTROPIC RECEPTORS Some of these receptors have the binding site on the extracellular side and then the ligand must arrive from the outside, in other, the latter is placed inside the cell, and the ligand is produced in the cytoplasm. The random match between channel and ligand leads to the formation of the receptor-ligand complex, and is therefore, entirely analogous to the formation of the enzyme substrate complex. In both cases, in fact, the binding takes place thanks to the weak forces, and leads to a conformational change of the entire protein and to the opening of the channel. There are also other important similarities: The formation of the binding site-ligand complex takes place in a totally random way and therefore dependent on the concentration of the ligand in the extra- / intracellular space. This phenomenon is of great importance in chemical synapses.

4 IONOTROPIC RECEPTORS In many cases, the binding site can accommodate several molecules from the ligand. These substances may have endogenous origin (adjustment molecules) or exogenous (drugs). They can be further classified into: agonists competitive antagonist Non-competitive antagonists Once formed, the receptor-ligand complex is reversible, with the ligand that can be rereleased into the environment or degraded

5 IONOTROPIC RECEPTORS Some receptors are provided with additional binding sites that recognize different molecules from the ligand. In analogy with the phenomenon of enzymatic allosteric regulation, also in this case the site is defined allosteric and can be occupied by allosteric modulators, classified as: allosteric activator allosteric inhibitor Even in these cases, the origin of allosteric modulators, can be endogenous or exogenous.

6 IONOTROPIC RECEPTORS Also for the ionotropic receptors, there is the general rule of ion channels about the refractoriness of time between an opening and the next.

7 IONOTROPIC RECEPTORS Compared to voltage-gated ion channels, ionotropic receptors have a much greater structural variability. They can be comprised of 3 to 5 subunits, and are normally classified into classes, including both receptor regulated by neurotransmitters (released in the synaptic junctions), and by second messengers (contained within the cells, eg. Ca + and camp). Among the various classes, the following are the most important for the nervous system: 1. Ionotropic receptors of the superfamily of nicotinic receptors 2. Ionotropic glutamate receptors 3. Ionotropic receptors of cyclic nucleotides 4. Ionotropic receptors regulated by Ca + 5. Ionotropic receptors regulated by other modes (light, mechanical tension, etc...)

8 Ionotropic receptors of the superfamily of nicotinic receptors They are all made up of 5 main sub-units, with both terminals in the extracellular environment. For this reason they are also called pentameric receptors. These sub-units may be the same or different from each other and combined in various ways depending on the receptor in question. Each subunit contains four regions of alpha-helical transmembrane (M1-M4), connected to each other by short loops that create aqueous pore on the region M3. In addition, on the extra-cellular side, these receptors have one or more binding sites for neurotransmitters, and / or for allosteric modulators. As regards the type of transported ions, their level of selectivity is relatively low, and then carrying cations (Na +, K + and Ca +) or anions (Cl-) The ionotropic nicotinic receptors have a wide variety of isoforms and therefore a very high number of receptor subtypes.

9 Recettori ionotropi della superfamiglia dei recettori nicotinici Nicotinic receptors ACh Serotonine GABA-A Glycin Excitatory - depolarization Inhibitors - hyperpolarization

10 ACh The nicotinic receptors for the acetylcholine (ACh), get their name because of their potent agonist nicotine. Since they were the first ionotropic receptors to be studied and classified, they serve as a paradigm for all pentameric ionotropic receptors.

11 ACh The structure and functionality of the receptors for ACh are different depending on the tissue in which they are expressed. It is possible in fact to have: muscle receptors (with subunits - alpha, beta, gamma, delta and epsilon) neuronal receptors (with subunits - alpha and beta)

12 Serotonine The 5-HT3 receptor is a channel-receptor activated by ligand serotonin (5-hydroxytryptamine) that allows the flow of Na + and K +; It has a similar structure to the ones of the nicotinic cholinergic receptors, with 5 sub-units called 5-HT3a-e. The subunit 5-HT3a is the bearer of the binding site with serotonin and is thus present in each receptor in combination with the other subunits. The binding of serotonin on two receptor sites determines the opening of the channel with consequent depolarization. These receptors are located on the parasympathetic endings in the gastrointestinal tract and also in the splanchnic and vagal afferents. In the central nervous system (CNS), on the other hand, there is a high density of 5-HT3 receptors in the nucleus of the solitary tract and the area postrema (where the vomiting center), but also in the nucleus accumbens, amygdala, hippocampus, entorhinal cortex and frontal) The 5-HT3 receptors in the gastrointestinal tract and in the CNS are involved in the emetic response and form the anatomical basis for antiemetic properties of 5-HT 3 receptor antagonists.

13 GABA-A The GABA-A is the receptor for the gammaaminobutyric acid (GABA) which is the most important inhibitory neurotransmitter in the brain. GABA is the endogenous agonist of the receptor and binds to the binding site, mediating an allosteric modification that does open the channel to anions, especially Cl-. The channel is formed by different subunits, slightly different depending on the nervous district in which is located, but in general there are: 2 α subunits 2 β subunits 1 γ subunit

14 GABA-A There are many agonists and antagonists of GABA A receptor, which bind to different subunits in different specific binding sites, dedicated to them, the most important include: Benzodiazepines (anxiolytics) Barbiturates (sedatives, hypnotics) Steroids (hormones derived from cortisol) Ethanol (anxiolytic-like effect) Picrotoxin (blocker)

15 GABA-A Even in the case of the GABA-A, the various sub-units can present different isoforms. In particular we have 6 isoforms to the alpha unit, 4 for the beta isoforms, isoforms and 4 for the gamma. These isoforms are combined with each other in various ways giving origin to a wide repertoire of receptor subtypes expressed in a specific way in the various districts of the CNS

16 Glycin The receptor for the glycine (GlyR) is a receptor similar to the Gaba-A, in fact, the same agonists and antagonists also act on this type of receptor. However, this receptor in the brain is present in quantities far lower than the GABA-A., With a limited distribution in the brainstem and spinal cord, and in addition, in the retina. In the embryonic stage of the glycine receptor it is composed of 5 alpha subunit. In the adult SNC It is composed of 3 alpha subunit (4 isoforms) and 2 beta. The 5 receptor subunits are assembled to form a center channel permeable to Cl- ion. Their disruption causes a disease called as Hyperekplexia (excessive alarm reaction).

17 IONOTROPIC RECEPTORS Compared to voltage-gated ion channels, ionotropic receptors have a much greater structural variability. They can be comprised of 3 to 5 subunits, and are normally classified into classes, including both receptor regulated by neurotransmitters (released in the synaptic junctions), and by second messengers (contained within the cells, eg. Ca + and camp). Among the various classes, the following are the most important for the nervous system: 1. Ionotropic receptors of the superfamily of nicotinic receptors 2. Ionotropic glutamate receptors 3. Ionotropic receptors of cyclic nucleotides 4. Ionotropic receptors regulated by Ca + 5. Ionotropic receptors regulated by other modes (light, mechanical tension, etc...)

18 Ionotropic glutamate receptors This class of receptors includes 3 different types, called AMPA, kainate and NMDA, which despite being activated by the same amino acid neurotransmitter glutamate (glutamic acid), are different from each other both in structure and in function. Their names depend on the specific agonists which allowed their identification: AMPA = alpha-amino-3-hydroxy-5-methyl-4-isoxa-zol-propionic acid Kainate = kainic acid NMDA = N-methyl-D-aspartate Their importance stems from the fact that glutamate is the absolute most common neurotransmitter in the brain and is the main agent of the excitatory neurotransmission. These receptors mediate important functions such as synaptic plasticity (learning and memory), but if over-stimulated (stroke, epilepsy) may kick off neurotoxicity processes leading to cell death. It is therefore possible that these receptors have a role at the base of neurodegenerative diseases.

19 Ionotropic glutamate receptors Regarding their specificity, the aqueous pores of AMPA and kainate receptors have a lower specificity and allow the passage of both K + that of Na +, and to a lesser amount of Ca2 +. In contrast the NMDA receptor has a marked specificity for Ca2 + and much lower for the other cations. NMDA AMPA, Kainate

20 Ionotropic glutamate receptors From the structural point of view, these receptors are composed of 4 or 5 sub-main units, characterized by a common basic organization of the polypeptide chain, with the N-terminal end in the extracellular environment and the C-terminal end in the intracellular environment. Each subunit contains always 3 trans-membrane regions (M1, M3 and M4) and a loop (M2) located in the intracellular side the purpose of which is to control the permeability of the aqueous pore.

21 Ionotropic glutamate receptors The specificity and the importance of these receptors is indirectly confirmed by the multiplicity of the types of subunits that make them up. These sub-units can be divided into groups based on similarities in their amino acid sequences

22 Ionotropic glutamate receptors Glutammate receptors AMPA Kainate NMDA GluR1 GluR3 GluR3 GluR4 KA1, 2 GluR5 GluR6 GluR7 NR1 NR2A,B C,D NR3A,B Excitatory

23 AMPA The AMPA receptors are the ionotropic more present in our brain and the main mediators of fast excitatory transmission. They consist of four sub-units (GluR1-4), of which 2 are always GluR1. Each type of sub-units is presented with several variations and subtypes that originate from post-transcriptional modifications of RNA, such as: alternative splicing RNA editing Or by post-translational modifications of the polypeptide chain, such as phosphorylation.

24 AMPA In the face of only 4 genes that encode for the 4 basic types of sub-ampa units, the RNA molecules undergo various modifications which entails the production of a vast number of types and subtypes of receptor, each of which has its own functional characteristics and may be expressed differentially in the various parts of the brain. In order to study this vast possibilities of these receptor, crops in vitro are used, or animal models in which the genes coding for the AMPA are selectively mutated. Through these studies it could be shown that AMPA antagonists are able to prevent cell death following a stroke, inhibiting the excessive stimulation of AMPA. In conclusion, the AMPA play a vital role in the stimulation of electric and indirect regulation of neuronal cell death.

25 Kainato The kainate (kainic acid) is a convulsant agent, that besides being an agonist of AMPA receptors, is able to activate the channels that have this molecule as selective agonist, and are precisely denominated channels of kainate. These channels are generally formed by a complex of tetrameric 5 KA1-2 and iglur5-7 possible sub-units that fit together. These subunits may variously be combined between them, but the presence of GluR5 or GluR6 is stable in order to obtain a receptor that, when activated selectively by the agonist, is able to generate a current intense cationic modulation. Both of these two subunits can exist in two different variants: in the sequence of amino acids in the porechannel wall, are located both the variable with arginine which makes this little permeable to calcium receptor, both the glutamine residues that make it highly permeable to sodium and calcium.

26 Kainate Less abundant in the CNS of AMPA receptors, these receptors are mainly expressed in the striatum, in the reticular nucleus of the thalamus, hypothalamus, in the deep layers of the cerebral cortex, the layer of granular cells of the cerebellum, in the dentate gyrus and in the glossy layer of ' CA3 hippocampal area. The kainate receptors appear to play an important role in the development and plasticity of the CNS since at birth they vary in number and in the expression of the different sub-units and are also involved in long-term potentiation (LTP). The KA receptors coexist with other receptor subtypes in postsynaptic level. Activation of these receptors also seem to modulate the release of GABA in hypothalamus and hippocampus.

27 NMDA NMDA receptors have a much slower kinetics (in the order of hundreds of milliseconds) of the AMPA and KA receptors and are highly permeable to calcium. As a rule, they act together with AMPA receptors and Ka, but their specific characteristics are the basis of the involvement of these in all higher cognitive processes but also diseases such as psychosis or schizophrenia. They are typically composed of four sub-units, each of which presents variation caused exclusively by alternative splicing. The different subunits include: NR1, NR2A-D (common) and NR3A-B, NR4 (inhibitory). The NR1 subunit is always present in all of the NMDA receptors. The peculiarity of this subunit is given by the sequence of amino acids which delimits the wall of the pore-channel where there are asparagine sites that make this receptor highly permeable to calcium and give this receptor other properties such as the one to bind magnesium ions, which in the non-opening of the receptor, are binded within the aqueous pore blocking completely the functionality. The NR1 subunit is ubiquitously in all brain regions while NR2A-D subunits are present preferentially in the cortex, in hippocampus and cerebellum.

28 NMDA The NMDA receptors have two different binding sites of the main endogenous ligands that respectively bind the L-glutamate, L-aspartate, L- omocistinate and chinolinate, and as coactivators ligands: glycine, D-serine and D-alanine. As said, within the channel, there is a site for the binding of magnesium ions. The activation of the NMDA receptor can take place only if at the same time both the glutamate and the glycine interact in their binding sites, in addition to these conditions, however, are necessary additional contingencies.

29 NMDA In total there are three, then, the situations necessary for the channel activation: 1. Receptor binding ligand or glutamate agonists. 2. Glycine present on the second binding site 3. Removal of Mg ++ ions. This is possible because on the postsynaptic membrane near the receptor there are also present rapid kinetic AMPA receptors which, when activated, lead to a rapid entry of calcium (less) and sodium that induce a rapid depolarization of the membrane which promotes the removal of magnesium ions, making possible the functioning of the receptor. The freeze due to magnesium can also be removed by endogenous polyamines: spermidine and spermine; at low and high concentrations, respectively, enhance and inhibit receptor activity. Along with these substances, also ketamine and phencyclidine act as non-competitive antagonists, causing phenomena similar to the positive symptoms of schizophrenia. In conclusion, the NMDA receptors are subjected to both the ligand control and the membrane potential control.

30 IONOTROPIC RECEPTORS Compared to voltage-gated ion channels, ionotropic receptors have a much greater structural variability. They can be comprised of 3 to 5 subunits, and are normally classified into classes, including both receptor regulated by neurotransmitters (released in the synaptic junctions), and by second messengers (contained within the cells, eg. Ca + and camp). Among the various classes, the following are the most important for the nervous system: 1. Ionotropic receptors of the superfamily of nicotinic receptors 2. Ionotropic glutamate receptors 3. Ionotropic receptors of cyclic nucleotides 4. Ionotropic receptors regulated by Ca + 5. Ionotropic receptors regulated by other modes (light, mechanical tension, etc...)

31 Ionotropic receptors of cyclic nucleotides Receptors of cyclic nucleotides CNG HCN Cyclic Nucleotide- Gated Hyperpolarization and Cyclic Nucleotide- Gated Excitatory

32 CNG HCN They are receptors of cyclic nucleotides (camp and cgmp), in tetrameric structure in which two subunits are homologues. Both the ends amino and carboxyl, are intracellular. These receptors are composed of 6 transmembrane helices. Moreover, there is a sequence of amino acids that enters from the extracytoplasmic side, it crosses the membrane partly folds in on itself, and flows out from the same side of the membrane (P LOOP). This amino acid sequence with the S6 helix participates in the formation of pore-channel wall. The binding of the ligands occurs on the intracellular side. On the other hand, camp and cgmp are formed within the cell and their binding site has to be intracellular. Receptors in the HCN S4 region contains a voltage sensor completely analogous to those of the voltage-regulated receptor. The receptors of the cyclic nucleotides perform important tasks in the photoreceptors of the retina, in the olfactory sensory epithelium cells, over that, in various districts of the CNS and P. HCN receptors are also particularly important for the regulation of the heartbeat.

33 IONOTROPIC RECEPTORS Compared to voltage-gated ion channels, ionotropic receptors have a much greater structural variability. They can be comprised of 3 to 5 subunits, and are normally classified into classes, including both receptor regulated by neurotransmitters (released in the synaptic junctions), and by second messengers (contained within the cells, eg. Ca + and camp). Among the various classes, the following are the most important for the nervous system: 1. Ionotropic receptors of the superfamily of nicotinic receptors 2. Ionotropic glutamate receptors 3. Ionotropic receptors of cyclic nucleotides 4. Ionotropic receptors regulated by Ca + 5. Ionotropic receptors regulated by other modes (light, mechanical tension, etc...)

34 Ionotropic receptors regulated by Ca + It is a diverse group of ionotropic receptors whose aqueous pore is selective for K + and opens when the intracellular binding site, binds a Ca2 + ion. Their molecular structure is similar to that of the channels regulated by voltage, they are then formed by tetrameric complexes with 6 transmembrane domains. The α1 subunit is the responsible for all electrophysiological and pharmacological properties of these channels since the pore and the voltage sensor are located on it. The 4th segment represents the potential sensor and between the 5th and 6th α-helix there is the binding site that characterizes the ion selectivity. There are 9 isoforms of the α1 subunit.

35 Ionotropic receptors regulated by Ca + The receptors regulated by Ca2 +, are normally classified according to their permeability for K + ions, specifically we can have: High permeability (BK receptors) Intermediate permeability (IK receptors) Low permeability (SK receptors)

36 IONOTROPIC RECEPTORS Compared to voltage-gated ion channels, ionotropic receptors have a much greater structural variability. They can be comprised of 3 to 5 subunits, and are normally classified into classes, including both receptor regulated by neurotransmitters (released in the synaptic junctions), and by second messengers (contained within the cells, eg. Ca + and camp). Among the various classes, the following are the most important for the nervous system: 1. Ionotropic receptors of the superfamily of nicotinic receptors 2. Ionotropic glutamate receptors 3. Ionotropic receptors of cyclic nucleotides 4. Ionotropic receptors regulated by Ca + 5. Ionotropic receptors regulated by other modes (light, mechanical tension, etc...)

37 Ionotropic receptors regulated by other modes (light, mechanical tension, etc...) This type of receptor, commonly also called mechanoreceptors, is activated by a variety of pressure stimulus such as those relating to tactile perception, auditory balance and the positioning in space of our body. They also participates, in the homeostatic regulation in the kidneys, and the control of blood pressure. As for the receptors in our skin, these act as pressure detectors due to a direct contact with the extracellular matrix (connective tissue) and the proteins of the receptor complex.

38 Ionotropic receptors regulated by other modes (light, mechanical tension, etc...) A second type of mechanoreceptors regard those places in the organ of Corti, in 'inner ear. These receptors constitute the final step of a series of vibrations that depart from the eardrum, and following transmission through the ossicular chain, up to the oval window, finally come to move the liquid contained in the organ of Corti. These shifts move a membrane called the tectorial membrane, which is in direct contact with the hair cells (stereocilia) which depending of their movement, open and close their channels, sensitive to Na + or Ca2 +.

39 Metabotropic receptors They are receptors that respond to the arrival of extracellular ligands and give way to a cascade of metabolic processes within the cytoplasm. They are very heterogeneous from a structural point of view, and can be comprised of a single polypeptide chain, or of 2 different sub-units which in the presence of the ligand, unite with each other. From the functional point of view we can classify them into two large families: Receptors linked to enzyme activity Receptors bound to G protein

40 Receptors linked to enzyme activity The receptors related to enzymatic activities are very diverse from a structural point of view, although the most famous and important are normally made up of two polypeptide chains, which as a result of ligand binding, unite their zones in the extracellular domain in a dimer. The formation of extra-cellular receptor dimer, allows the intracellular protein portions to initiate an enzymatic activity, which in most cases is a protein kinase. The kinases consists in the phosphorylation of other proteins or in the self-phosphorylation of the receptor. Depending on which amino acid is phosphorylated the name of the receptors could change in: Tyrosine kinase Histidine kinase Serine / threonine kinase The phosphorylation operated by these receptors is typically the first step in a series of important metabolic processes that follow the arrival of ligands such as growth factors (skin, neurotrophic, fibroblasts and platelets) or hormones (insulin).

41 Receptors bound to G protein The metabotropic receptors are membraneproteins bound on the cytoplasmic side to G proteins (GPCR), and their activation starts a process called signal transduction.

42 Receptors bound to G protein G proteins are proteins with GTPase activity formed by 3 subunit α, β, γ. In the inactive form, αlpha subunit binds GDP and is associated closely to the complex formed by the β and γ subunits. When activated by the interaction with the receptor, the α subunit undergoes a conformational change that causes its detachment from the βγ complex, and the exchange of GDP, already bound to it, with a molecule of GTP. In this way such subunits become active and can interact with target proteins (primary effector) The subunit αlpha not remains in the active conformation for a long time, by virtue of its intrinsic GTPase activity, which rapidly detaches the terminal phosphate group from GTP, transforming it into GDP. This activity provides a cellular response strictly dependent on the activation of the metabotropic receptor.

43 Ligand Signal transduction Metabotropic receptor Enzyme - inactive GDP G protein

44 Signal transduction Bond of the Ligand with the receptor first messanger conformational change in the receptor that leads him to bind with the receptor

45 Signal transduction Following binding to the receptor, the protein expels GDP and binds instead to a GTP GDP GTP

46 Signal transduction The presence of GTP instead of GDP cause the detachment of the protein from the receptor, and the division into two subunits G-alpha-GTP and the heterodimer Beta-gamma. By the way, they remains anchored to the plasma membrane and are free to move nearby the receptor area.

47 Signal transduction The complex G-alpga-GTP meet an enzyme that will act as the primary effector in the transduction process. The enzyme will start using the energy derived from the hydrolysis of GTP to GDP to produce new molecules that will act as second messengers to go to activate secondary effector Likewise, also the betagamma complex will bind to other primary effectors to start the production of other metabolic processes

48 After the hydrolysis of GTP to GDP, the alpha complex detaches from the primary effector ending the production of second messengers. Once back free in the cytoplasm the alpha complex can meet a beta-gamma complex free and reform the original G protein. Signal transduction

49 Signal transduction

50

51 Receptors GPCRs possess seven transmembrane domains to α-helix, a binding site for the neurotransmitter place deep in the center of the portion that faces the extracellular and intracellular domain that makes contact with G protein And through the interaction with these proteins that metabotropic receptors exert their effects.

52 Receptors Most classical neurotransmitters active both ionotropic and metabotropic receptors. Each of these transmitters can induce both rapid responses (msec), such as excitatory postsynaptic potentials or inhibitors, either to slow onset and long-lasting responses. This possibility provides to the nervous system the ability to process the information in time and to modulate the response to various environmental requirements. This allows metabotropic receptors to play a key role in the CNS and PNS. Their importance is further confirmed by the high number of genes (in the order of thousands) that code for these proteins, 350 of which are receptors for neurotransmitters or other known ligands while 150 have not yet had a positive identification, and are therefore called orphan receptors.

53 Receptors In general, GPCRs can be grouped into three families: The family A, is by far the most numerous and includes most of the receptors for the monoamines and neuropeptides. Family B consists of the secretin receptor, glucagon and calcitonin. The C family consists mainly of metabotropic glutamate receptors and receptors sensitive to Ca2 + An activated receptor ligand can activate many copies of G proteins with a cascading effect that amplifies the extracellular signal.

54 G protein and first effector There are several classes of G proteins with specificity both in the target and in the activity. Gs, Gi, and Gq are the most important.

55 Second messanger The second messengers are responsible for the induction of new cellular activities: camp activates protein kinase A (PKA), calcium ions activate, together with diacylglycerol, protein kinase C (PKC) and, through interaction with calmodulin, another regulatory protein, the calcium-calmodulin dependent protein kinase (CAMK ). All of these kinases, cascading, phosphorylate many protein targets within the cell, changing the activity. Another important function of PKA is the activation of CREB transcription factor that activates the transcription of genes that encode proteins that once synthesized, may bind to ion channels, enzymes and / or structural proteins, modifying the activity.