GPCR Block A

Question 1

What do you expect to happen with the amplitude of K+ current through BK channels if you block voltage gated Ca2+ channels? Explain why.

Answer 1

K+ current would decrease as the K+ channel is activated by Ca2+, thus if you block the Ca2+ channels, there is less Ca2+ to activate the K+ channels.

Question 3

What are the main differences between nicotinic and muscarinic Acetylcholine receptors?

Answer 3

The primary difference is the way in which the channels function after binding ACh. For example, when a nicotinic ACh receptor binds ACh it opens to allow the flow of ions to occur, also known as ionotropic, thus allowing depolarisation to take place. In the case of muscarinic ACh receptors they are metabotropic receptors. Meaning that when ACh binds to these receptors it activates the G-protein. These G-proteins can vary and have different cellular responses depending on which G-protein is activated and what proteins these G-proteins act.

Question 4

Why stimulation of muscarinic ACh receptors in pacemaker cells in SA node slows down heart rate? Describe the chain of events.

Answer 4

As pacemaker cells in the heart express M2 ACh receptors when ACh binds to these receptors it activates the Gαi/o signalling pathway. This pathway begins with the inhibition of adenylyl cyclase, reduction of intracellular levels of cAMP. As Gbetagamma subunit opens inward rectifying potassium channels. This causes an increased hyperpolarisation as K+ is rushing out of the cell. As K+ is positive with the loss of these ions cause the decrease in membrane potential and thus it is more difficult to raise the cell membrane potential to that of a Na+ depolarisation.

Question 4

Make a list of common serine/treonine kinases and their activators.

Answer 4

1. Protein kinase A (PKA) - activated by cAMP (Gs pathway)
2. Protein kinase C (PKC) - activated by diacylglycerol (Gαq¬/ Gα11 pathway)
3. Protein kinase G (PKG) - activated by cGMP
4. Ca2+/ calmodulin dependent kinases (camK family) - activated by calcium/ calmodulin

Question 5

Describe the steps of GPCR signalling common for all GPCRs.

Answer 5

1. At rest G-protein forms a heterotrimeric complex (alpha, beta and gamma subunits; alpha subunit is bound to GDP)
2. Ligand binding to GPCR causes conformational change that allows the receptor to interact with G-protein and function as a guanine nucleotide exchange factor (GEF).
3. GEF exchanges GDP bound to alpha-subunit to GTP
4. GTP-bound alpha-subunit and betagamma-subunit dissociate from the receptor and each other
5. Alpha and betagamma-subunits can now interact with their targets
6. GTP on alpha-subunit is hydrolysed to DGP, alpha and betagamma subunits bind to GPCR.

Question 5

What cellular enzyme is activated by transducin? In what types of cells?

Answer 5

The cellular enzyme phosphodiesterase is activated by transducin, this occurs in photoreceptor cells only.

Question 6

Activation of which Galpha-proteins leads to generation of Diacylglycerol in a cell? What other second messengers are produced in the same signalling pathway?

Answer 6

Gαq and Gα11 lead to generation of diacylglycerol in a cell. Other second messengers within this pathway include inositol trisphosphate.

Question 6

What effect does cAMP have on the open probability of “ZERO” splice variant of BK channel? What is the mechanism of this effect?

Answer 6

cAMP Increases phosophokinase A, which adds phosphate group to serine residue. cAMP thus increases open probability of channel as increase in phosphorylation increases open probability.

Question 8

What are the main levels of ion channels regulation by GPCRs?

Answer 8

1. Direct modulation of channel function by binding of a G protein
2. Modulation by synthesis of a second messenger that binds to the channel
3. Modulation by second-messenger-mediated activation of a kinase that phosphorylates the channel, or phosphatase that dephosphorylates the channel.

Question 8

Which signalling pathway is activated by stimulation of β-adrenergic GPCRs in the heart?

Answer 8

Gαs pathway

Question 10

Make a list of the main second messengers generated by stimulation of GPCRs. Which of them are lipids?

Answer 10

Cyclic AMP
Cyclic GMP
Inositol 1,4,5-triphosphate
Ca2+
Phophatidylinositol (PIP2)
Diacylglycerol (DAG)
Arachidonic acid

Question 10

What is the resting membrane potential of vertebrate photoreceptor cells? How does it change in response to a flash of light?

Answer 10

-40mV in the dark. When a flash of light occurs the resting membrane potential will actually decrease.

Question 11

Why high levels of intracellular cAMP in cardiomyocytes promote faster and stronger heart contractions? Explain the mechanism.

Answer 11

Increase in cAMP activates protein kinase A
PKA phosphorylates voltage gated Ca2+ channels
Phosphorylated CaV
Channels have higher open probability and therefore conduct more Ca2+ ions into the cardiomyocytes, therefore resulting in faster stronger contractions.

Question 13

Give an example of an ion channel directly modulated by a G-protein. Which type of G-proteins regulates ion channels directly?

Answer 13

The G protein-coupled inwardly-rectifying potassium channels (GIRKs) are a family of inward-rectifier K+ channels which are activated by binding of Gbetagamma subunit. Gbetagamma subunit that interacts with GIRK channels only comes from Gαi/o pathway.

Question 14

Which neurotransmitter is released from photoreceptor cells in the dark? What types of receptors it activates on bipolar neurons? How activation of these receptors affects membrane potential of bipolar neurons?

Answer 14

Glutamate is released in the dark due to consta depolarisation.

Question 16

What signal activates Rhodopsin? What is the function of Rhodopsin?

Answer 16

A light photon will activate rhodopsin which changes 11-cis retinal to 11-trans retinal. The change in shape causes a conformational change in the opsin protein that binds it leading to an activated form of rhodopsin. As the opsin has undergone a conformational change it can interact with transducin, which causes transducin to get rid of GDP and take up a GTP. This then causes an activation of PDE, which is phosphodiesterase. The GTP tranducin binds with PDE, which activates it. There is then a conversion of cGMP to GMP via PDE. Sodium ion channels are embedded into the plasma membrane which causes a constant stream of Na+ in which causes a constant depolarisation and release of glutamate neurotransmitter. Sodium channels need cGMP to activate it, if this is not there then the channel will close. Therefore when PDE changes cGMP to GMP no more sodium channels will be activated and the Na+ no longer cause a constant depolarisation, as such, there is no an absence of glutamate release and the body interprets this as light being received.

Question 17

What is the general function of GPCRs? Which physiological processes are regulated by GPCRs (list 5 examples)?

Answer 17

Main function of GPCRs is to transduce extracellular stimuli into intracellular signals.
Photo transduction - light receptors (opsins)
Sense of smell - receptors of the olfactory epithelium
Sense of taste - receptors of sweet, bitter, and amino acid (Umami)
Regulation of brain activity - serotonin, dopamine, GABAb, glutamate
Metabolism - glucagon, ghrelin

Question 18

3. List all known G(alpha) subunits. Which of them regulate production of cAMP?

Answer 18

Gαs - activates plasma membrane adenylyl cyclases, increasing cellular cyclic AMP (cAMP), which stimulates phosphorylation of target proteins by cAMP-dependent protein kinase (PKA).
Gαi/ Gαo - inhibit most adenylyl cyclases, decreasing cellular cAMP.
Gαq¬/ Gα11 - activate phospholipase Cbeta (PLCbeta) which generates several second messengers (inositol trisphosphate, diacylglycerol) and activates Ca2+ signalling pathway and protein kinase C (PKC).
Gα12, Gα13 - activate RhoA pathway (regulation of cytoskeleton)
Gαtransducin - activates cyclic GMP (cGMP) phosphodiesterase that cleaves and depletes cytoplasmic cGMP (in retina only).
Gαgustducin - activates cAMP phosphodiesterase that cleaves and depletes cAMP (taste receptors)
Gbetagamma - directly activates G protein-coupled inwardly-rectifying potassium channels.

Question 19

How the activity of CNG channels in photoreceptor cells is related to the activity of phosphodiesterase?

Answer 19

CNG stands for cyclic nucleotide gated channels (permeable to Na+ and Ca2+). It is regulated by cGMP. When cGMP is present the CNG channel remains open. Phosphodiesterase, when activated, is responsible for the change of cGMP to GMP. When PDE is converting cGMP to GMP it causes a decrease in cGMP levels inside the cell. Eventually the cGMP won’t be sufficient to bind to the CNG channels and the channels will close. Therefore high PDE productivity means low CNG being activated.