31st Oct - Ion Channels and GPCR signalling

Question 1

Why are ion channels useful membrane transporters?

Answer 1

Can conduct >1000000 ions per second
Can be voltage gated or ligand gated
Show amazing selectivity

Question 2

What are the two major forms of modulation of G proteins?

Answer 2

Indirect - i.e. via 2nd messengers

Direct - i.e. via direct G protein binding

Question 3

Outline the Beta1 adrenoceptors indirect modulation of cardiac l-type calcium channels

Answer 3

Cardiac B1 adrenoceptor –> Gs –> AC –> PKA –> cardiac L ytpe calcium channel –> increase in force of contraction

Question 4

Outline the M1 receptors indirect modulation of neuronal K+ channels

Answer 4

M1 receptor –> Gq –> PL-C –> PIP2 –> DAG and IP3 –> PKC and Ca2+ –> Neuronal K+ channel (KCNQ 2/3) –> Enhanced neuronal excitability

Question 5

Outline the direct modulation of cardiac GIRK 1/4 by M2 muscarinic receptors

Answer 5

M2 Muscarinic receptor –> Gi –> Cardiac K-ACh Inward Rectifier K+ Channel (GIRK1/4) –> Slowing of heart rate

Question 6

Outline the direct modulation of N, P/Q type calcium channels by Gi

Answer 6

NA/GABA/5-HT/DA/Opiods/Cannabinoids –> Gi –> N, P/Q type calcium channels –> Pre-synaptic inhibition

Question 7

What are the main functions of calcium?

Answer 7

Muscle Contraction
Neurotransmission and hormone release
Gene Expression

Question 8

What is the key method of controlling calcium flux?

Answer 8

Modulating VGCC

Question 9

Outline the experiment performed by Dunlap and Fischbach in 1981

Answer 9

Whole cell calcium currents recorded with voltage clamp of chick sensory neurons in the presence/absence of 100µM NA
With NA the change in current was reduced, than the negative control –> VGCC can be modulated by GPCRs

Question 10

Give an example of a pre-synaptic GPCR that can inhibit VGCC

Answer 10

Mu, Kappa and delta opiod receptors
Cannabinoid receptor 1 and 2
GABAb
D2,3,4
Serotonin 1A
Alpha 2 adrenergic receptor
M2, M4 ACh receptor
Adeonsine 1 receptor

Question 11

How does pertussis toxin affect the cell?

Answer 11

It blocks the modulation of VGCC by GPCRs by inactivating the alpha subunits of Gi/Go

Question 12

How was the mechanism of GPCR modulation of VGCC elucidated by Forscher, Oxford and Schultz (1986)?

Answer 12

Used a patch clamp in the whole cell configuration with the cell in a bath containing the agonist –> record inhibitory effect

Used a patch clamp in the cell attached configuration (to isolate the channels under the membrane patch) with the cell in a bath containing the agonist –> no effect

Therefore it is not a diffusible second messenger i.e. G alpha which transmits the modulation of the ion channel, as there is no effect from the ion channels within the patch

Question 13

Outline the experiment performed by Ikeda (1996)

Answer 13

Held Rat sympathetic neurons at -80mV
Added a GTP analogue –> amplitude of the calcium current was reduced
Injected beta gamma subunit –> huge reduction in the amplitude of the calcium current
Alpha subunit –> no change compared to control
Beta subunit alone –> no change compared to control
Gamma subunit alone –> no change compared to control

Question 14

Outline the structure of a VGCC

Answer 14

alpha 1 subunit contains 4 repeats of 6TM domain structure which form a pore in the membrane
alpha 2 subunit extracellular
Intracellular Beta subunit
Delta subunit - transmembrane which generally couples to the alpha 2 subunit
Gamma subunit - transmembrane

Question 15

What can be used to pharmacologically to inhibit Q and N-type calcium channels?

Answer 15

omega conotoxin

Question 16

What can be used to pharmacologically to inhibit P-type calcium channels?

Answer 16

omega agatoxin

Question 17

What can be used pharmacologically to inhibit L-type calcium channels?

Answer 17

DHPs

Question 18

What can be used pharmacologically to inhibit T-type calcium channels?

Answer 18

Mibefradil and flumarizine

Question 19

What are the types of calcium channels that are high voltage activated?

Answer 19

P-type
Q-type
N-type
L-type
R-type

Question 20

Outline the experiment performed by Bourinet (1996) which shows what type of calcium channels are inhibited by each G protein

Answer 20

Used DAMGO (mu opiod receptor agonist (Gi)) on varying types of calcium channel and monitored effects

Cav2.2 (with alpha2delta and beta subunits) (N-type) –> large inhibition
Cav2.1 (with alpha2delta and beta subunits) –> small inhibition
Cav1.2 (L-type) (with alpha2delta and beta subunits) –> no inhibition

He then extended this to so that he measured the effect of DAMGO on:
alpha 1A alone, found major inhibition about 50-65%
alpha 1A, with alpha 2 delta and beta, found only about 20% inhibition

–> Hypothesis: Beta gamma subunit is competing with the beta subunit of the calcium channel and the beta gamma subunit of the g protein

Question 21

Outline Zamponi’s 1997 experiment identified the binding site for the beta gamma subunits to the VGCC

Answer 21

Used the amino acid sequence, and identified regions of consensus between the N, P and Q type channels whic are regulated by the G protein. Identified QXXER binding motif in the linker region between domains 1 and 2

Question 22

Outline the molecular mechanism of VGCC inhibition by g proteins

Answer 22

Beta gamma subunits alter the voltage sensitivity of VGCC, meaning that a greater depolarisation is required for them to open

Question 23

Outline the experiment used to elucidate the mechanism of GPCR inhibition on VGCC

Answer 23

A patch clamp in the single channel recording configuration was used, to measure the open probability against the potential the cell is held at.

Found that channels fell into 2 populations:
Willing which opened easily
Reluctant which require a greater depolarisation for the channel to open