Lecture 5 - Neuronal cell signalling

Question 1

What is brought about by neurotransmitters interacting with ligand gated ion channels and G protein coupled receptors on the post synaptic membrane?

Answer 1

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

Question 2

Aside from binding to receptors/channels on the post synaptic memebrane how else can neurotransmitters modulate excitability?

Answer 2

By activating presynaptic autoreceptors (either ligand gated or G protein coupled)

Question 3

What is the function of GPCRs?

Answer 3

regulate the funciton of voltage-gated and ligand gated ion channels

Question 4

What can the regulation of voltage gated and ligand gated ion channels result in ?

Answer 4

1. Trigger excitation/inhibition
   - post synaptic potential
   - primary excitation of sensory cells (receptor potential)
2. Modulate excitability
   - syanptic transmission
   - adaptation of sensory neurons

Question 5

What do GPCRs rely on to mediate responses?

Answer 5

Not a channel so must rely on signalling events to mediate resonses

Question 6

When GPCRs are found on the presynaptic membrane what is their function?

Answer 6

GPCRs on the presynaptic membrane modulate neurotransmitter release

Question 7

What are the pre and post synaptic targets of GPCR regulated ion channels?

Answer 7

Pre
-voltage gated calcium channels that coupled action potential to neurotransmitter release
Post
-ligand gated and voltage gated ion channels

Question 8

What are signalling events in the post synaptic cell triggered by?

Answer 8

• 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

Question 9

What is the process of GPCR signalling?

Answer 9

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

Question 10

What are the Gα effectors?

Answer 10

Enzymes that generate second messengers through the binding of a ligand to a GPCR (activates it)

Question 11

What are the three categories of Gα effectors?

Answer 11

Gs units
Gq units
Gi units

Question 12

What is the process of signalling from the Gs type of Gα effector?

Answer 12

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

Question 13

What is the process of signalling from the Gq type of Gα effector?

Answer 13

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

Question 14

What is the process of signalling from the Gi type of Gα effector?

Answer 14

1 - inhibits Adenyl cyclase
2- reduces cAMP level
3- leading to reduced levels of pKA and a decrease in protein phosphorylation

Question 15

What are the different targets of Gα and Gβγ?

Answer 15

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

Question 16

How was it shown whether intracellular signalling was mediated by the second messenger or by the Gβγ unit?

Answer 16

-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

Question 17

How does adrenaline function as a neurotransmitter and how was this shown experimentally?

Answer 17

-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

Question 18

How was it shown that Gβγ subunits activate a K+ channel in response to ACh?

Answer 18

-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

Question 19

How is ion channel activity modulated by GPCR signalling activity?

Answer 19

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

Question 20

What are GIRKs?

Answer 20

Presynaptic K+ channnels

-regulate membrane excitability

Question 21

How do GPCRs regulate presynaptic excitabiltiy?

Answer 21

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

Question 22

How can glutatmanergic neurons modulate neurotransmitter release?

Answer 22

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

Question 23

How can GABAergic neurons modulate neurotransmitter?

Answer 23

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

Question 24

What is involved in Post synaptic modulation?

Answer 24

• 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

Question 25

What are the most crucial 2nd messengers?

Answer 25

Ca2+ ions

Question 26

When are Ca2+ signals prominent in the post synaptic cell?

Answer 26

When exposed to gluatamatergic inputs from the pre-synaptic neuron

Question 27

What are the extracellular sources of Ca2+? (ie how does it get into the post synaptic neuronal cell)

Answer 27

Ligand gated channel - e.g. NMDA
Voltage gated calcium channel - post synaptic L type VGCC
Store operated channels

Question 28

What is the intracellular source of Ca2+?

Answer 28

The ER

Question 29

What are the release channels for Ca2+ on the ER, and what are they activated by

Answer 29

IP3 receptors - activated by increased IP3 levels (From the Gq metabotropic glutamate receptor)

Ryanodine receptors - activated by Ca2+ coming in through the ligand gated channel (NMDA) and VGCC

Question 30

Describe the whole process of CA2+ enterance in the post synaptic cell

Answer 30

• GPCR anatgonised by glutamate.
• IP3 activated (via Gq)
• IP3 activates IP3R and Ca2+ released from ER
• The same (and other) Ca2+ ions activate the ryanodine receptor andeven more Ca2+ is realeased form the ER
• Meanwhile, the lgc, VGCC (and SOC) are antagonised, e.g. by glutamate and open to allow CA2+ from outside the cell in
• In cases where the interal store of calcium in the ER is depleted, a signal is released from an ER bound Ca2+ sensor to the SOC, which is triggered to allow more Ca2+ into the cell.

Question 31

What channels/pumps/exchange systems remove ca2+ from the post synaptic neuron?

Answer 31

PMCA - actively pumps Ca2+ out of the cell
NCX - Na+/Ca2+ exchange system
Serca - sarcoplasmic endoplasmic reticulum Ca2+ ATPase (pumps Ca2+ back into ER)

Question 32

How is excitation in a sensory cell mediated? Give an example

Answer 32

cyclic nucleotide gated channels - they are ion channels directed to open by a 2nd messenger
E.g. cGMP gated Na+ channels mediate changes in activity of photoreceptor cells

Question 33

What is one other hypothesized way that when the ER is depleted in get’s more CA2+ into the cell?

Answer 33

It releases STMI which signals to TRPC channels to allow more CA2+ in.

NB some TRPC channels ingluenced by Gq signalling (e.g. IP3)
ie. store operated and modulated by 2nd messengers