Lecture 5 - Neuronal cell signalling Flashcards
What is brought about by neurotransmitters interacting with ligand gated ion channels and G protein coupled receptors on the post synaptic membrane?
Ligand gated ion channels
-NT binding opens ligand gated ion channels to mediate direct excitation or inhibition of the postsynaptic cell
G protein coupled receptors
-NT interaction initiates intracellular signalling events to exert effects on neurotransmission
Aside from binding to receptors/channels on the post synaptic memebrane how else can neurotransmitters modulate excitability?
By activating presynaptic autoreceptors (either ligand gated or G protein coupled)
What is the function of GPCRs?
regulate the funciton of voltage-gated and ligand gated ion channels
What can the regulation of voltage gated and ligand gated ion channels result in ?
- Trigger excitation/inhibition
- post synaptic potential
- primary excitation of sensory cells (receptor potential) - Modulate excitability
- syanptic transmission
- adaptation of sensory neurons
What do GPCRs rely on to mediate responses?
Not a channel so must rely on signalling events to mediate resonses
When GPCRs are found on the presynaptic membrane what is their function?
GPCRs on the presynaptic membrane modulate neurotransmitter release
What are the pre and post synaptic targets of GPCR regulated ion channels?
Pre
-voltage gated calcium channels that coupled action potential to neurotransmitter release
Post
-ligand gated and voltage gated ion channels
What are signalling events in the post synaptic cell triggered by?
- GPCRs: such as metabotropic neurotransmitter receptors that generate cAMP, cGMP, DAG and IP3
- Ca2+ signalling downstream of VGCC, mediated by ligand gated Ca2+ channels and GPCRs
What is the process of GPCR signalling?
1-Binding of a ligand to a GPCR activates teh receptor, causing the α subunit to exchange GDP for GTP
2-this leads to dissociation of the α-GTP complex from the βγ subunits and the receptor
3- the α subunits interact and influence the activity of an effector protein
4 - this is terminated by GTPase activity of the αsubunits (enhanced by GAPS)
5 - GDP bound α subunit reunites with the βγ subunits
What are the Gα effectors?
Enzymes that generate second messengers through the binding of a ligand to a GPCR (activates it)
What are the three categories of Gα effectors?
Gs units
Gq units
Gi units
What is the process of signalling from the Gs type of Gα effector?
1-activated by β adrenergic GPCR
2-interacts with and activated Adenyl cyclase (membrane bound enzyme)
3-convert ATP to cAMP
4- cAMP interacts with pKA (protein kinase A) and activates it
5- which can then phosphorylate various substrates e.g. ion channels and modulate the activity of those ion channels
What is the process of signalling from the Gq type of Gα effector?
Gq type e.g. mGluR
1- acitvates phospholipase (enzyme on the membrane)
2- breaks down lipids in the membrane to Diacyglycerol and the second messenger IP3
3- IP3 triggers intracellular calcium release and Ca2+ activates Ca2+ dependent kinases e.g. CAMkinaseII
4- Diacyglycerol activates Protein kinase C to alter the phosphorylation of various proteins including ion channels
5-This increases protein phosphorylateion and activation calcium binding proteins e.g. calmodulin
What is the process of signalling from the Gi type of Gα effector?
1 - inhibits Adenyl cyclase
2- reduces cAMP level
3- leading to reduced levels of pKA and a decrease in protein phosphorylation
What are the different targets of Gα and Gβγ?
Gα
Adenyl cyclase
Guanylyl cyclase
Phospholipase C
->Influence ion channels via generation of second messengers cAMP, cGMP, DAP and IP3
Gβγ
direct binding of Gβγ subunits influences ion channels
How was it shown whether intracellular signalling was mediated by the second messenger or by the Gβγ unit?
-Used a Cell-attached patch technique [Giga-ohm seal prevents movement of neurotransmitter between extracellular medium and solution in the pipette]
-recorded Ca2+ current in neuron that recieved a depolarisation
1-applied neurotransmitter (adrenaline) to the outside of the patch pipette and showed that more recpetors were open and for longer
-> if the NT is applied outside the pipette the only way it can get inside is via an intracellular messenger which can regulate the activity of the VGCC
How does adrenaline function as a neurotransmitter and how was this shown experimentally?
-activates Gs coupled receptor, to generate cAMP (second messenger)
Shown
1- Intracellular injection of cAMP elicits the same effect as the neurotransmitter
2- PKA phosphorylates channel to increase open probability and time
How was it shown that Gβγ subunits activate a K+ channel in response to ACh?
-used a detatched membrane patch inside-out, for single channel recordings
-applied ACh inside the patch pipette and pulled away from the rest of the cell (so it can no longer communicate with the cytoplasmic region of the cell)
-ACh activates muscarinic ACh receptor (GPCR) and also activates a ligand gated ion channel (nitotinamin ligand gated ion channel - present at high quantities in the neuromuscular junction)
activity (K+ channel activity, GDP activity) under these conditions showed that all the necessary molecules for signalling reside in that patch of membrane
How is ion channel activity modulated by GPCR signalling activity?
Gq coupled receptor
- PLC
- then increases levels of IP3 and DAG
- IP3 increase leads to increased Ca2+ which as a second messenger can activate other channels
- DAG increases the affinity of VGCC
Gs
- increases cAMP
- increases PKA levels
- consequently increases the affinity of VGCC
Gi/o coupled receptor
- βγ sububnits activate thrugh potassium cahnnels, GIRKs
- α subunits decrease levels of cAMP
- decreases activity of of VGCC
What are GIRKs?
Presynaptic K+ channnels
-regulate membrane excitability
How do GPCRs regulate presynaptic excitabiltiy?
Though VGCC that couple AP to NT realse and presynaptic K+ channels (GIRKs) that regulate membrane excitabilty
Inhibitition
-decrease in NT release due to inhibiton of VGCC and activation of K+ channels via Gi/o coupled receptors
Facilitation
-increase NT release due to activation of VGCCs by Gs-coupled receptors
How can glutatmanergic neurons modulate neurotransmitter release?
Glutamate acting on the presynaptic nerve terminal opens presynaptic voltage gated calcium ion channels
- results in an increase/prolongation of the calcium current,
- inhibits potassium channels
- prolongs the AP and leads to an increase in the release of theneurotransmitter
How can GABAergic neurons modulate neurotransmitter?
GABA binds to G protein coupled receptor (Gi/o coupled)
- leads to a decrease cAMP
- reduced activity of VGCC in the presynaptic membrane
- leading to a smaller calcium current and consequently less neurotransmitter release
- and the excitatory post synaptic potential is reduced
GABA can also activate potassium channels which would shorten the duration of the AP and cause less NT to be released
What is involved in Post synaptic modulation?
- Glutamanergic synapses release glutamate activating AMPA receptors allowing an influx an Na+ depolarising the membrane
- when the membrane is depolarised and in the presence of extrancellular glutamate the activated ligand gated glutatmate receptor NMDA allows an influx Na+ and Ca2+ (as the blocking Mg2+ ions that blocked the pore moves away during depolarisation)
- this triggers intracellular signalling and protein kinase C
- this leads to the activation of of calmodulin kinase II which phosphorylates AMPA receptors inside the cell increasing their trafficking to the cell surface
- AMPA receptors on the cell surface can also be phosphorylated to affect their gating (open longer and more sensitive)
This way, the next time the presynaptic neuron fires an AP, the post synaptic response will be quicker as there are more AMPA receptors
