Lecture 12: Metabotropic Neurotransmitter Receptors Flashcards
What can metabotropic receptor activation lead to?
can lead to delayed, sustained PSPs – leads the opening of a potassium-selective ion channel
What type of responses do metabotropic receptors produce?
slow, but sustained and diverse responses
What are some consequences that arise from employing intracellular cascades for signalling?
- delay: latency of ~100ms - 1000ms between reception of the signal and effects on the cell
- persistence: effects have sustained duration (from seconds to 10-15 minutes), and outlast the presence of neurotransmitter within the synaptic cleft
- diverse responses: he same metabotropic receptor can simultaneously trigger a number of different effects in the same cell (ie. more than just a change in Vm)
Can metabotropic receptor responses differ even when they share neurotransmitters?
yes – same neurotransmitter can have opposite responses by acting on different subtypes of metabotropic receptor
What does the muscarinic M1 receptor do?
triggers depolarization of the cell when activated by ACh by inhibiting a K+ conductance
What does the muscarinic M2 receptor do?
triggers hyperpolarization of the cell when activated by ACh by enhancing a K+ conductance
What does the adrenergic 𝛼1A receptor do?
(norepinephrine receptor) triggers vasoconstriction by enhancing smooth muscle contraction
What does the adrenergic 𝛽2 receptor do?
(norepinephrine receptor) triggers vasodilation by reducing smooth muscle contraction)
What family of proteins do metabotropic receptors belong to?
all belong to GPCR superfamily of proteins
NOTE: all metabotropic receptors are GPCRs, but not all GPCRs are neurotransmitter receptors
What is another name for metabotropic neurotransmitter receptors?
g-protein coupled receptors (GPCRs) – reflects their association with g-proteins
What are g-proteins?
intracellular signalling proteins
What are g-proteins?
intracellular signalling proteins
What are some ligands that GPCRs detect?
- endogenous ligands (these include neurotransmitters and hormones)
- olfactory and gustatory ligands, and light
What are some structural features of all GPCRs?
- 7 transmembrane (TM) domains, organized in a precise sequence
- no ion-permeable transmembrane pore
- intracellular G-protein binding site
What are some structural features of all metabotropic receptors?
- all features of GPCRs (3)
- and extracellular ligand binding site
What are the two aspects of GPCR signalling you need to know for this course?
- how activating a GPCR leads to activation of g-proteins – and how g-proteins are inactivated
- how activated g-proteins lead to biochemical signalling cascades – and how this differs between different g-protein subtypes
What do g-proteins bind?
guanosine phosphates
What do g-proteins work closely with?
they are the intracellular partners of GPCRs – act as intracellular molecular switches, transducing signals detected by GPCRs into intracellular biochemical pathways
Where are g-proteins found?
in all eukaryotic cells
What are g-proteins named for?
their ability to bind the signalling molecule GTP, or its inactive relative GDP
How are g-proteins activated?
- inactive g-protein contains a GDP molecule in its ‘guanosine’ binding site
- activation of a GPCR (bound to neurotransmitter or agonist) catalyzes activation of the g-protein by promoting exchange of GDP for a new GTP molecule
What are the 2 types of g-proteins?
monomeric g-protein
heterotrimeric g-protein
Which type of g-protein is used in neurotransmitter synaptic signalling?
heterotrimeric G-proteins – most (if not all) GPCRs that respond to neurotransmitter bind to heterotrimeric G-proteins
Which type of g-protein is more common?
heterotrimeric G-proteins
What are monomeric g-proteins also known as?
- small GTPases
- Ras superfamily
What are monomeric g-proteins important for?
cell growth responses
- Ras and other monomeric G-proteins are important for cell growth and division
- in neurons, they have roles in dendrite and axonal growth, synapse formation, and remodelling (but not neurotransmitter signalling)
What are monomeric G-proteins activated by?
GTP binding – BUT they do not directly associate with GPCRs
GPCRs activate monomeric G-proteins by using adaptor proteins and/or guanosine exchange factor proteins (GEFs)
What is the structure of heterotrimeric G-proteins?
consist of three (different) subunits – when the complex is inactive (GDP bound to α subunit), the three subunits assemble together on a GPCR
- α subunit
- β and γ subunit
What do α subunits of heterotrimeric G-proteins do?
bind GDP or GTP
What do α subunits of heterotrimeric G-proteins do when activated (GTP-bound)?
they diffuse away from GPCR and affect activity of other intracellular proteins
What do β and γ subunits of heterotrimeric G-proteins do?
functional unit – when α subunit binds a new GTP and becomes activated, β and γ separate from α and can also affect certain intracellular proteins
What do GTPase activating proteins (GAPs) do?
promote inactivation of G-proteins by catalyzing hydrolysis of GTP (back to GDP)
How many kinds of g-protein can each GPCR bind to?
can typically only bind to (and thus activate) one kind of G-protein
this means that more than one GPCR is often able to activate the same kind of G-protein
How many kinds of g-protein can each GPCR bind to?
can typically only bind to (and thus activate) one kind of G-protein
this means that more than one GPCR is often able to activate the same kind of G-protein
How do g-proteins mediate signal transduction cascades?
through the production of second messengers
What are the 3 key ideas to remember for g-protein-mediated signal transduction?
- cascades
- amplification
- protein kinase
What are cascades?
multi-step pathways where one step catalyzes the production/activation of a distinct molecule/enzyme for the next step
What is amplification?
at each step of a cascade, one activated molecule can make many more of the units in the next step
What are protein kinases?
enzymes which catalyze the addition of phosphate groups onto other proteins
G-protein-mediated Signal Transduction
ligand (first messenger) + receptor → (amplification) → g-protein → enzyme → (amplification) → second messenger → protein kinase → (amplification) → phosphate transferred to target proteins
What are messengers?
ligands – molecules whose function is to bind specific receptors or enzymes within a signalling pathway, and switch that protein on or off
What are regulators?
molecules that bind to enzymes and alter the ongoing activity of those enzyme
What are effectors?
molecules that actually make a direct change to physiology of cell, rather than continuing the cascade
The definition of messengers and regulators can, and does overlap a lot. When are you more likely to use the term ‘regulator’?
if the targeted enzyme is affecting effector proteins, not making a new type of messenger molecule
What are the 3 types of messengers in cascades?
- first messengers
- second messengers
- third messengers
What are first messengers?
extracellular ligands that bind to surface receptors (neurotransmitters, hormones, neuropeptides)
What are some functions of first messengers? (2)
- fast PSPs
- produce second messengers
What are second messengers?
intracellular molecules that regulate enzymes and channels (Ca2+, cyclic nucleotides, DAG, IP3)
this level can also include transducer proteins (ie. G-proteins, calmodulin), but they are not technically classified as second messengers because they are proteins (although they have some common features)
What are some functions of second messengers? (5)
- slow PSPs
- modify ion channels
- local structural changes
- local metabolic changes
- produce 3rd messengers
What are third messengers?
molecules that enter the nucleus and interact with DNA (DNA-binding proteins/transcription (co-)factors)
What are some functions of third messengers? (4)
- gene expression
- protein synthesis
- global structural changes
- global metabolic changes
What is the difference between a regulator and a second messenger?
- second messenger turns it on
- regulator can modify it
How are Gα subunits classified?
into five groups based on the intracellular pathways (targets) they activate
What are the 5 groups of Gα subunits?
- G αs (stimulatory)
- G αi/o (inhibitory)
- G αq (‘queer’):
- G αt (transducin)
- G α12/13 (rho-mediated)
What does the G αs (stimulatory) subunit group do?
activate adenylate cyclase (AC) and enhance cAMP signalling pathway
What does the G αi/o (inhibitory) subunit group do?
inhibit adenylate cyclase (AC) and reduce cAMP signalling pathway
What does the G αq (‘queer’) subunit group do?
activate phospholipase C (PLC) and enhance IP3/DAG signalling pathway, including ↑[Ca2+]
What does the G αt (transducin) subunit group do?
activate cGMP phosphodiesterase (PDE) and reduce cGMP signalling pathway
What does the G α12/13 (rho-mediated)) subunit group do?
interact with GEFs and members of Ras family of monomeric GTPases
Remember: transduction cascades also activate mechanisms that can limit/turn themselves off.
-
Cascade #1: ‘Stimulatory’ G αs Signalling Pathway
How does this pathway signal?
by increasing cAMP levels
Cascade #1: ‘Stimulatory’ G αs Signalling Pathway
- Gαs-GTP binds to AC (primary enzyme)
- AC synthesizes cAMP (second messenger) from ATP
- cAMP regulates cyclic nucleotide gated channels (effector proteins)
- cAMP binds and activates PKA (cascade enzyme)
- PKA phosphorylates many protein targets (effector proteins) – ie. Ca channels, K channels, GluARs, SNAREs, active zone proteins, other kinases and transcription factors
- PKA targets phosphatases (PP), which remove phosphate groups so that effector proteins are not activated forever
- PKA targets phosphodiesterases (PDE), which break down cAMP
What is adenylyl/adenylate cyclase (AC)?
membrane-bound enzyme present in all cells that makes cAMP from ATP
What are cyclic nucleotide gated channels?
intracellularly gated K+/cation channels
Can transduction cascades turn themselves off?
yes – they activate mechanisms that can limit/turn themselves off
Cascade #2: ‘Inhibitory’ G αi Signalling Pathway
How does this pathway signal?
by decreasing cAMP levels
Cascade #2: ‘Inhibitory’ G αi Signalling Pathway
- Gαi-GTP binds to AC (primary enzyme)
- AC cannot synthesize cAMP (second messenger)
(opposite of cascade #1)
How does Gαt signal?
by activating PDE instead of inhibiting AC
Gαt is a closely related subunit to Gαi
Cascade #3: G αq Signalling Pathway
How does this pathway signal?
by increasing DAG/IP3 levels
Cascade #3: G αq Signalling Pathway
- Gαq-GTP binds to phospholipase C or PLC (primary enzyme)
- PLC synthesizes IP3 and DAG (second messengers)
- IP3 regulates IP3-gated Ca2+ channels, which releases Ca2+ (second messenger)
- Ca2+ enhances PKC, and targets intracellularly (effector proteins) – ie. ion channels, signalling proteins (calmodulin, vesicle proteins, transcription factors)
- DAG binds and activates PKC
- PKC phosphorylates many protein targets (effector proteins) – ie. some ion channels, SNAREs and vesicle proteins, other kinases and transcription factors (cascade)
- phosphatases (PPs) turn off the signal
- block PKC
- block IP3
- remove phosphates from PKC targets
What are some roles that Ca2+ can play in signal transduction?
- second messenger – activating calmodulin, which then activates other regulator enzymes
- regulator –directly binding and activating various regulator enzymes, including PKC and some forms of AC
- direct effector – ie. ie. controlling synaptotagmin and gating Kca channel activity
What is one of several mechanisms through which cells can elevate their [Ca2+]i?
g-protein signalling
What is the source of the increases in [Ca2+]i during g-protein signalling?
extracellular or intracellular – from ER stores
Does an increase in [Ca2+]i during g-protein signalling affect Vm?
when the release of Ca2+ ER stores is intracellular, there is no direct effect on Vm
What mechanisms do intracellular calcium ions act through in biochemical cascades?
- directly interacting with effector proteins or enzymes
- binding to transducer proteins which then activate other enzymes/proteins
What is the reason why a neurotransmitter can have opposite effects depending on which metabotropic receptor it binds to?
because different subtypes couple to different alpha subunits
What does the stimulatory alpha subunit subtype (Gαs) do to smooth muscle? To heart muscle?
- causes smooth muscle relaxation – smooth muscle contractions are inhibited by cAMP, though they are stimulated by Ca2+
- causes increase in heart muscle contraction
What are some roles of G-βγ subunits?
- important signalling roles in neuronal transduction pathways – once unbound from α subunit, βγ complex can act as a signalling molecule and mediate a variety of intracellular effects, including important effects on electrical signals
- can bind and directly trigger opening of certain ion channels (typically K+ selective), changing Vm
- can modify VG-Ca channel properties (typically raises their activation threshold – inhibits activation)
- sometimes alter the activity of regulator enzymes
What is the Neuron Doctrine?
direction of information flow through the nervous system is unidirectional (axon AP → neurotransmitter → dendritic EPSP)
Does chemical synaptic transmission fit the Neuron Doctrine?
yes…
BUT you can find receptors on the presynaptic terminal in certain circumstances
What are the 3 circumstances in which receptors are found on the presynaptic terminal?
- axoaxonic synapses
- autoreceptors
- retrograde signals
What are axoaxonic synapses?
synapses that make direct (one-way) contact between axon terminals
Where are axoaxonic synapses found?
in many different neuronal circuits across different animal species (but are not as common as axodendritic or axosomatic synapses)
What is the function of axoaxonic synapses? Do they use ionotropic or metabotropic receptors?
- some are efficient inhibitory synapses using inhibitory ionotropic receptors
- most are modulatory synapses that signal through metabotropic receptors – these can affect both VG-K and VG-Ca channels, and also lead to phosphorylation of active zone proteins such as SNAREs and docking proteins
When axoaxonic synapses are modulatory and signal through metabotropic receptors, what can this affect?
can affect both VG-K and VG-Ca channels, and also lead to phosphorylation of active zone proteins such as SNAREs and docking proteins
What are autoreceptors?
metabotropic receptors on a presynaptic terminal that respond to the same neurotransmitter released by that terminal
What are autoreceptors usually (but not always)?
GPCRs coupled to Gi/o subtypes, and produce negative feedback on neurotransmitter release
ie. ACh-releasing terminals possess muscarinic ACh receptors (mAChRs)
ie. GABA releasing terminals express GABABRs
What subunit signalling contributes to inhibition of neurotransmitter release by autoreceptors?
both α and βγ signalling
What are retrograde signals?
ligands that are synthesized and released from the postsynaptic cell (or a glial cell)
What is sensitive to retrograde signals?
sometimes presynaptic receptors are sensitive to these
What are true examples of retrograde synaptic signalling?
some presynaptic metabotropic receptors
Are retrograde messengers hydrophobic or hydrophilic?
hydrophobic
When are retrograde messengers synthesized?
synthesized on demand, but synthesis is often triggered in response to metabotropic receptor activation by conventional neurotransmitters (ie. by increase in cAMP or Ca2+)
What are cannabinoid receptors (CBs or CBRs)?
principal targets of cannabinoid drugs (THC, CBD)
- in CNS, CBRs are GPCRs that are typically found on presynaptic terminals, and couple to Gαi/o subtypes
the best described examples of presynaptic receptors that receive ‘retrograde message’
What are endocannabinoids?
endogenous molecules that activate cannabinoid receptors
two best known: anandamide, 2-AG
What is THC?
cannabinoid agonist
When are endocannabinoids synthesized?
synthesized by postsynaptic neurons in response to a rise in intracellular Ca2+ through VG Ca2+ channels, NMDARs, and/or αq mediated signalling
What do endocannabinoids do once they’re synthesized?
diffuse through postsynaptic membrane and bind the CBRs located on the presynaptic terminal
How do metabotropic neurotransmitter receptors signal?
by initiating intracellular biochemical signalling cascades (rather than by directly opening and closing ion channels)
Do metabotropic responses trigger effects on membrane potential?
yes – by coupling intracellular signals (second messengers) to internally gated ion channel opening and closing
What are metabotropic receptors? How do they act?
g-protein coupled receptors that mediate distinct cellular effects by activating different heterotrimeric g-protein-mediated cascades
