31st Oct - Ion Channels and GPCR signalling Flashcards
Why are ion channels useful membrane transporters?
Can conduct >1000000 ions per second
Can be voltage gated or ligand gated
Show amazing selectivity
What are the two major forms of modulation of G proteins?
Indirect - i.e. via 2nd messengers
Direct - i.e. via direct G protein binding
Outline the Beta1 adrenoceptors indirect modulation of cardiac l-type calcium channels
Cardiac B1 adrenoceptor –> Gs –> AC –> PKA –> cardiac L ytpe calcium channel –> increase in force of contraction
Outline the M1 receptors indirect modulation of neuronal K+ channels
M1 receptor –> Gq –> PL-C –> PIP2 –> DAG and IP3 –> PKC and Ca2+ –> Neuronal K+ channel (KCNQ 2/3) –> Enhanced neuronal excitability
Outline the direct modulation of cardiac GIRK 1/4 by M2 muscarinic receptors
M2 Muscarinic receptor –> Gi –> Cardiac K-ACh Inward Rectifier K+ Channel (GIRK1/4) –> Slowing of heart rate
Outline the direct modulation of N, P/Q type calcium channels by Gi
NA/GABA/5-HT/DA/Opiods/Cannabinoids –> Gi –> N, P/Q type calcium channels –> Pre-synaptic inhibition
What are the main functions of calcium?
Muscle Contraction
Neurotransmission and hormone release
Gene Expression
What is the key method of controlling calcium flux?
Modulating VGCC
Outline the experiment performed by Dunlap and Fischbach in 1981
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
Give an example of a pre-synaptic GPCR that can inhibit VGCC
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
How does pertussis toxin affect the cell?
It blocks the modulation of VGCC by GPCRs by inactivating the alpha subunits of Gi/Go
How was the mechanism of GPCR modulation of VGCC elucidated by Forscher, Oxford and Schultz (1986)?
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
Outline the experiment performed by Ikeda (1996)
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
Outline the structure of a VGCC
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
What can be used to pharmacologically to inhibit Q and N-type calcium channels?
omega conotoxin
What can be used to pharmacologically to inhibit P-type calcium channels?
omega agatoxin
What can be used pharmacologically to inhibit L-type calcium channels?
DHPs
What can be used pharmacologically to inhibit T-type calcium channels?
Mibefradil and flumarizine
What are the types of calcium channels that are high voltage activated?
P-type Q-type N-type L-type R-type
Outline the experiment performed by Bourinet (1996) which shows what type of calcium channels are inhibited by each G protein
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
Outline Zamponi’s 1997 experiment identified the binding site for the beta gamma subunits to the VGCC
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
Outline the molecular mechanism of VGCC inhibition by g proteins
Beta gamma subunits alter the voltage sensitivity of VGCC, meaning that a greater depolarisation is required for them to open
Outline the experiment used to elucidate the mechanism of GPCR inhibition on VGCC
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
