Ionotropic and metabotropic receptors - Università di Roma LUMSA · Recettori ionotropi della...

Ionotropic and metabotropic

receptorsLESSON NR. 10 - PSYCHOBIOLOGY

• 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.

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- / intra-

cellular space. This phenomenon is of great importance

in chemical synapses.

• 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 re-

released into the environment or degraded


• 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.


Also for the ionotropic receptors, there is the

general rule of ion channels about the

refractoriness of time between an opening

and the next.


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 ...)


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.

Nicotinic receptors

ACh Serotonine GABA-A

Recettori ionotropi della superfamiglia dei recettori nicotinici

Glycin

Excitatory - depolarization Inhibitors - hyperpolarization

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.

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)

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.

GABA-A

The GABA-A is the receptor for the gamma-

aminobutyric 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

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)

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

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).







cAMP).










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.

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.

AMPA, KainateNMDA


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 intra-

cellular 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.


The specificity and the importance of these receptors is

indirectly confirmed by the multiplicity of the types of sub-

units that make them up.

These sub-units can be divided into groups based on

similarities in their amino acid sequences


Glutammate receptors

AMPA Kainate NMDA

GluR1

GluR3

GluR3

GluR4

KA1, 2

GluR5

GluR6

GluR7

NR1

NR2A,B

C,D

NR3A,B

Excitatory


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.

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.

AMPA

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 pore-

channel 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.

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.

Kainate

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.

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.

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.







cAMP).










Receptors of cyclic nucleotides

CNG HCN

Cyclic Nucleotide-

Gated

Hyperpolarization and

Cyclic Nucleotide-

Gated

Excitatory

Ionotropic receptors of cyclic nucleotides

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.

CNG HCN







cAMP).










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.

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)

Ionotropic receptors regulated by Ca +







cAMP).










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.

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 +.

Ionotropic receptors regulated by other modes (light, mechanical tension, etc ...)

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

• 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).

• Receptors bound to G protein

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

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.

• Receptors bound to G protein

Signal transduction

Ligand

Metabotropic receptor

G protein

Enzyme - inactive

GDP

Bond of the Ligand with the receptor – first messanger

conformational change in the receptor that leads him

to bind with the receptor

Signal transduction

Following binding to the receptor, the protein expels

GDP and binds instead to a GTP

GDP

GTP

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.

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 beta-

gamma complex will

bind to other primary

effectors to start the

production of other

metabolic processes

Signal transduction

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

Signal transduction

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.

• Receptors

• 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.

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.

• Receptors

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.

• G protein and first effector

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.

• Second messanger

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