Receptors and Channels Flashcards
1
Q
What does transmembrane mean?
A
Spans the membrane
2
Q
What is a channel?
A
A channel is a transmembrane protein that transports molecules from one side of the membrane to the other
3
Q
What are 3 essential functions of ion channels and an example of where these functions are used?
A
- Transport ions across membrane
>Stomach and salivary glands - Regulate membrane potentials
>Nerve and muscle cells - Ca2+ influx into the cytoplasm
>Secretion and muscle contraction
4
Q
What are the 2 factors ion channels are classified into sub groups based on?
A
- Gating mechanism – voltage or ligand
- Ion selectivity of the pore
5
Q
How many alpha-helices are transmembrane proteins usually made of in ion channels?
A
Transmembrane proteins made up of two or more ⍺-helices that cross the lipid bilayer.
6
Q
How many subunits are ion channels usually made of?
A
Made up of two – six subunits which usually surround the ‘pore’
7
Q
What defines the ion selectivity of an ion channel’s pore?
A
by the physical size of ‘filter’ and the charge of the amino acids lining the pore
8
Q
What is an alpha-helices?
A
A right hand-helix conformation
9
Q
What is a beta sheet?
A
Beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a sheet.
10
Q
What are subunits?
A
Single protein that forms with others to form protein complex
11
Q
What are transmembrane domains (TM)?
A
Protein that spans the width of the membrane from the extracellular to intracellular sides usually a helical shape
12
Q
What is a P-loop or a Pore?
A
Pocket where ion will bind
13
Q
What is useful about understanding the structure of different ion channels?
A
It reveals evolutionary relationships
14
Q
What is a primordial channel?
A
The first channel we know to exist, this acts as a basic structure of all ion channels.
15
Q
What 3 factors control the gate of a simple K+ channel?
A
- Membrane potential
- Mechanical stress
- Ligands binding to C-terminal
16
Q
In terms of transmembrane domains, what is conformation of a ion channel when the gate is a) closed b) open?
A
a) TMs are more tightly packed creating a ‘gate’
b) Upon ions flowing through the pore, the TMs move into an open conformation.
17
Q
What are the 2 main functions of voltage gated ion channels?
A
- Na+ and K+ create action potentials in excitable cells
- Ca2+ transported into cytoplasm where 2nd messenger elicits a cellular response
18
Q
What are 3 differences in structure that a voltage gated ion channel has that a simple one doesn’t?
A
- Additional helices S1 and S4 form a separate ‘voltage sensing domain’ lateral to the subunits
- Large polypeptides that extend into the cytoplasm
- Plugging mechanism
>Polypeptide strand can block the poor and stop ions entering
19
Q
What does TRP stand for?
A
Transient receptor potential channels
20
Q
How is a Transient receptor potential (TRP) channel similar and different to voltage gated ion channels?
A
Share common structural features with Voltage gated channels BUT evolved to sense chemicals and physical stimuli instead of membrane potential (e.g. hot and spicy food)
21
Q
How is a Ligand-gated ion channel similar and different to voltage gated ion channels?
A
Similar in structure to voltage-gated but controlled by the binding of a ligand
22
Q
What are 2 example of intracellular binding sites of ligand-gated ion channels?
A
- Calmodulin bound to C-terminal
- Cyclic nucleotide-binding domain
23
Q
What is the effect of Ca2+ binding to Calmodulin on a Ligand gated-ion channel?
A
If Ca2+ binds to calmodulin on the intracellular binding domain it provides negative feedback causing the channel to close.
24
Q
What are Ligand gated ion channels controlled by?
A
Either intracellular or extracellular ligands
25
Why does losing one ligand molecule bound to a ligand-gated ion channel cause a sharp drop in a concentration curve?
As Ligands must be bound to at least 3 out of 4 of the subunits to keep the gate open, if one ligand is lost the channel shuts quickly stopping the flow of ions.
26
Are a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels gated?
a) Simple channel = No gate
b) Voltage gated = Gate controlled by changes in electrical membrane potential
c) TRP = Gated
d) Ligand-gated = Gated, controlled by chemical transmitters either intra or extra cellular
27
How many subunits do a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels have?
a) Simple channel = 2
b) Voltage gated = 4
c) TRP = 4
d) Ligand-gated = 4
28
How many helices across the lipid bilayer do a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels have?
a) Simple channel = 2
b) Voltage gated = 6-24
c) TRP = 6
d) Ligand-gated = 6
29
Do all ion channels have a P-loop (central pore)
Yes
30
Do a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels have cytoplasmic anchores?
a) Simple channel = No as the channel goes straight through membrane
b) Voltage gated = Yes
c) TRP = Yes
d) Ligand-gated = Yes
31
Do a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels have voltage sensing domains?
a) Simple channel = No
b) Voltage gated = Yes, S1 and S4 helices form separate voltage sensing domain lateral to subunits
c) TRP = No
d) Ligand-gated = No
32
Do a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels have plugging mechanisms?
a) Simple channel = No
b) Voltage gated = Yes
c) TRP = Yes
d) Ligand-gated = No
33
What is an example function of a) Simple b) Voltage-gated c) Transient receptor potential channels (TRP) d) Ligand-gated ion channels?
a) Simple channel = Secretion/ absorption of fluids
b) Voltage gated = Excitable cells e.g. neurons, cardiac muscle
c) TRP = Hot and spicy taste
d) Ligand-gated = Olfaction (cAMP) and Muscle (Calmodulin)
34
How are extracellular Ligand-gated ion channels classified by?
Put into family defined by structure of the protein making up one of their subunits
35
What is the composition of a receptor in the Nicotinic receptor superfamily?
>Pentameric as has 5 subunits
>4 transmembrane domain regions in 1 subunit with pore (P-loop) in the middle.
>Extra cellular domain recognises neurotrasmitters.
36
Which subunit dictates the ion flow in a) Nicotinic receptor super family b) Glutamate receptor family c) ATP P2X family?
a) TM2 is the TM which dictates which ion flows through the channel
b) The half TM dictates which ion flows through the channel
c) TM2 is the TM which dictates which ion flows through the channel
37
What are 3 examples of receptors in the Nicotinic receptor superfamily (pentameric)?
Nicotinic acetylcholine receptor, serotonin receptors, GABAA receptors
38
What is the composition of a receptor in the Glutamate receptor family?
>Tetrameric as 4 subunits
>4 transmembrane domains per subunit,TM3 is a half subunit embedded in the membrane (alpha, beta and gamma)
>Has extra cellular ligand binding site (for Glutamate)
39
What is the composition of a receptor in the ATP P2X receptor family?
>Trimeric as has 3 subunits
>2 transmembrane domains per subunit
>Extra cellular binding site for ATP
40
What are 3 examples of receptors in the Glutamate receptor family?
AMPARs, NMDARs, KARs (Kainate receptors)
41
What is an example of a receptor in the ATP P2X receptor family?
P2X1-7
42
How can mutations in the transmembrane domain which dictates ion flow for an ion channel change its function?
A single amino acid change in these domains it will dictate which ion flows through the channel due to changing the charge of the protein
43
What is another name for a pentameric receptor?
Cys-loop type receptor
44
What is an example of a Cys-loop type receptor (pentameric) in muscle cells?
Nicotinic acetylcholine receptors (nAChRs)
45
What is the composition of Nicotinic acetylcholine receptors (nAChRs)?
>Pentameric, so is composed of 5 subunits
>Each subunit has 4 transmembrane domains (M1,M2,M3,M4) with external facing N domain and intracellular loop between M3 and M4
>M2 TM lines the poor and the charge makes it selective for specific ions
46
What is unique about Nicotinic acetylcholine receptors (nAChRs)?
Subunits can move to make more space for different ions to flow through the channel.
47
How do receptors in the same receptor family vary in different tissue?
Have different subunits so have different charges/ ion flow.
48
What is an advantage of the variety of subunit compositions within a receptor family and an example of this?
> Can target 1 subunit combination without effecting the other receptors.
> E.g. nAChR ⍺4 subunit is involved in reward pathways and nicotine addiction
49
What subunits can make up nAChRs in neurons?
α2 - 10 & β2 - 4 subunits can make different subunit compositions each with a different affinity depending on composition and location
50
What 2 subunits are abundantly expressed in neuronal nAChRs in the cortex and hippocampus, what agonists have a high affinity for them?
>Α4β2 subunits are abundantly expressed in the cortex and hippocampus and have high affinity to agonists nicotine and varenicline
51
How is addiction to smoking linked to A4B2 nAChRs in the hippocampus and cortex?
>Chronic exposure leads to receptor upregulation of A4B2 nAChRs causing addiction as more nicotine will be required to trigger a response.
52
What two polymorphisms in genes can make it easier to give up smoking?
Polymorphisms in subunit genes CHRNA4 (code for α4) and CHRNA6 (code for α6) make it easier to give up smoking.
53
What does ADNFLE stand for?
Autosomal dominant nocturnal frontal lobe epilepsy
54
How is Autosomal dominant nocturnal frontal lobe epilepsy caused?
>Mutations in M2 region of alpha4 neuronal nicotinic subunit causes use-dependent potentiation and delay in rising phase caused by a slow unblocking of closed receptors.
55
How does the delay in rising phase in mutated nicotinic receptors cause Autosomal dominant nocturnal frontal lobe epilepsy seizures?
Lag in rising phase (depolarisation) causes slow unblocking of closed mutant receptors, so when an input suddenly increases, a lot of acetylcholine is suddenly released overstimulated the CNS causing seizures.
56
What is the main neurotransmitter in the brain?
Glutamate (excitatory)
57
How is a Glutamate receptor similar and different in structure to a simple K+ channel
>Is tetrameric like a simple K channel, so 4 subunits surround a pore
>However usually 2 sets of the same 2 subunits which make up the 4 (is made up of 2 dimers). And contains an extra cellular ligand binding domain that is in a cleft that closes when occupied.
58
What is the structure of a simple K+ channel
>Tetrameric, 4 subunits surround a P-loop (pore)
>Each subunit is made up of 2 alpha helices (TMs)
59
What are 3 factors which contribute to the diversity of glutamate receptors?
Multiple genes, alternative splicing and RNA editing contribute to diversity
60
What are the 3 types Glutamate receptors?
1. AMPA = AMPA receptors mediate fast excitatory synaptic transmission in the central nervous system.
2. NMDA = N-methyl-D-aspartate receptor – involved in learning and memory – slower than other isoforms
3. Kainate = similar to AMPA but lesser role at synapses linked to Schizophrenia, depression and Huntington's
61
What are the 2 ways of processing RNA which causes glutamate receptor diversity?
RNA splicing and RNA editing
62
How does RNA splicing cause Glutamate receptor diversity?
>Alternative splicing of two exons in the primary transcript produces two protein isoforms with different domain in the extracellular loop; called flip and flop (different combinations of exons are spliced together, leading to the production of distinct mRNA isoforms from a single gene.)
>The two isoforms have different kinetic properties e.g. flop = faster desensitization rate and reduced current responses to glutamate than flip
63
How does RNA editing cause Glutamate receptor diversity and an example?
>Edit the TM lining the pore to have a different charge effecting the permeability to specific ions.
>E.g. If we edit the Q to R site in the M2 subunit of GluA2 receptor it won't become permeable to Ca2+
64
What is the effect on mice that lack the enzyme responsible for RNA editing for GluA2 at the Q/R region?
As all the GluA2 channels will be very permeable to Ca2+ (RNA editing reduces permeability to CA2+) they are prone to seizures and early death.
65
What is the function of NMDA (Glutamate) receptors and what does excess stimulation cause?
>Controls synaptic plasticity and mediates learning/ memory function
>Excess stimulation due to strokes leads to neuron death
66
How can ALS (Amyotrophic lateral sclerosis) cause damage to the body in terms of RNA editing of glutamate receptors?
Causes downregulation of GluA2 Q/R editing in the M2 TM causes increased Ca2+ permeability at AMPA receptors which increases glutamate activity causing damage due to glutamate excitotoxicity
67
How does glioblastoma (cancer of the brain) effect RNA editing, what is the effect of this and how can this be fixed?
>Decreased ADAR2 (RNA editing enzyme) activity correlated with increased malignancy due to Increase in Ca2+ effecting Akt pathway promoting proliferation and tumourigenesis.
>This was reversed when GluA2 Q/R was edited
68
How many molecules of ATP need to bind to a P2X receptor to activate it?
3 ATP molecules needed to open channel (as has 3 subunits)
69
What are 2 treatments that targeting the P2X7 receptor can be used for?
1. To stop inflammation
>As P2X7 activation of macrophages causes inflammation
2. To treat multiple sclerosis (MS)
>As P2X7 overexpression causes this.
70
What makes a receptor an attractive drug target in the clinic?
The diversity and specificity of the receptor
71
What is the a) extracellular ligand and b) example disease/ physiological condition for P2X (Trimeric) receptors?
a) ATP
b) P2X2 Hearing loss
P2X4 Pain
P2X7 inflammation, neurodegenerative disease
72
What is the a) extracellular ligand and b) example disease/ physiological condition for Glutamate (Tetrameric) receptors?
a) Glutamate
b) Excess NMDA in Stroke = neuron death
73
What is the a) extracellular ligand and b) example disease/ physiological condition for Cys-loop (pentameric) receptors?
a) Nicotinic acetylcholine, GABA, seratonin, Glycine
b) Epilepsy
74
What is an ionotropic receptor?
Direct exchange of ions through a pore in the ion channel
75
What is a metabotropic receptor?
Indirectly linked to ion channels through signal transduction mechanisms such as G proteins
76
How many super families of GCPCRs are there?
25 (most diverse type of receptor)
77
Why do receptors in the same family of GPCR bind different ligands?
Families share similar domains, but their amino acid sequence of their ligand binding sites can be different- so bind different ligands.
78
What activates a) ionotropic receptors b) GPCRs?
a) Mechanical stimuli, neurotransmitter, change in membrane potential
b) A wide range: lipids, photons, amino acids, nucleotides, proteins
79
What links helices in a GPCR?
Intracellular and extracellular loops.
80
What is the structure of a GPCR?
7 transmembrane domains in alpha helices (7 alpha helices), no subunits (just these 7 TMs)
81
Why does binding of a ligand allow a G-protein to bind to a GPCR?
Receptors become active when ligand binds, between TM5 and TM6 conformational change occurs causing opening of intracellular binding pocket for G-protein to bind.
82
What is an example GPCR found in platelets?
Protease-activated receptors (PAR)
83
What happens in terms of GPCR when the basil lumina is exposed due to an injury in 4 steps?
1. Basil Lumina exposure causes platelets to release ADP
2. ADP binds to GPCR on neighbouring platelets activating thrombin.
3. Thrombin cleaves N terminus of PAR ( Protease-activated receptors) causing activating of PARs.
4. Activation of PAR causes platelets to aggregate and form cross links making a clot.
84
What is the a) N terminus b) C terminus of a GPCR?
a) N terminus at extracellular side (ligand binding)
b) C terminus at intracellular side (G-protein binding)
85
What family do
Guanine nucleotide-binding proteins belong to?
Belong to the GTPase family
86
What is the function of Guanine nucleotide-binding proteins in GPCRs?
Act as molecular switches inside cell to transmit signals from extracellular stimuli to downstream effectors
87
What is the structure of Guanine nucleotide-binding proteins?
>Trimeric G-protein made up of 3 subunits:
1. Alpha
2. Beta
3. Gamma
88
What is the basic mechanism of action of a GPCR when a ligand binds in six steps?
1. Agonist binding to N terminus (extracellular)
2. TM 5 and 6 move apart allowing binding pocket (C terminus) to open on intracellular side
3. Alpha subunit binds to pocket
4. Alpha subunit bound to GDP in resting state, GDP gets displaced by GTP on binding, activated G-protein.
5. Alpha dissociates from membrane and from beta and gamma subunits, left with 3 active components.
6. Alpha moves through the membrane and acts on downstream effectors.
89
How is the duration of GPCR signalling regulated?
>Duration of signalling by activated trimeric G protein is regulated by rate of GTP hydrolysis by α subunit and the time the ligand is present.
90
How does a G-protein switch off?
To switch off, the alpha subunit hydrolyses GTP to GDP, so the rate limiting step is this hydrolysis.
91
How many G-protein families are there in humans and what makes them different?
There are seven different G-protein families in humans, their alpha subunits differ from each other.
92
What is the G-protein family that interacts with acetylcholine receptors, how many members are in it and their effect?
>Giα family has 7 members (all have Gi alpha subunit)
>Interacts with acetylcholine receptors causes negative feedback in neuronal synapses or decreases insulin from pancreas by inhibiting adenylyl cyclase.
93
What is the G-protein family that interacts with Rhodopsin receptors, how many members are in it and their effect?
>Gtα family has 2 members (all with Gt alpha subunits)
>Involved with Rhodopsin receptor responding to photons, allows us to see
94
What happens when an odour binds to an odour receptor in terms of G-proteins in 3 steps?
1. Odour binding to odour receptor causes activation of GPCR
2. Activates Golfα G-protein as GTP binds,
3. Activates Adenylyl cyclase which converts ATP to cAMP (cyclic AMP)
4. This activates cAMP gated channels allowing movement of ions into and out of the cell to elicit an action potential to the brain specific for that smell.
95
What are 2 factors that determine the response of a signal?
It is the specific subunit and cell type that determine the response using the same signalling as other cell types (The same signalling pathway could be used in a different cell type to elicit a different response)
96
What are 2 examples of effectors from GPCR pathways?
1) Enzymes
2) Ion channels
97
What is the function of an enzyme in a GPCR pathway?
Synthesise second messengers.
98
What are the 2 ways ion channels are regulated in GPCR pathways?
Gating is regulated either directly (β𝛾 subunits) or indirectly by 2nd messengers and their effectors
99
How does a GPCR indirectly activate an ion-channel and how is this different from an independent ionotropic receptor activating?
>Ligand binds to GCPR causing downstream signalling to open ion channel
>Is slower than ligand-gated ion channels being bound to directly. But the channel can stay open or closed for longer (minutes rather than milliseconds)
100
What are 3 examples of second messengers?
1. Hydrophobic lipids confined to the membrane in which they are generated
2. Small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)
3. Calcium ions (most basic and oldest second messenger)
101
What is a second messenger?
Second messengers are small molecules that carry signals inside cells
102
Why are second messenger systems necessary in GPCR signalling?
>Allows for amplification of the signal.
>A single ligand binding can activate many G-proteins which can activate many effectors which can generate many secondary messengers, which go on to activate many proteins
103
What effect does the Cholera Toxin from Vibrio Cholera bacterium have on GPCRs in 5 steps?
1. Activates Gsα subunit (stimulatory subunit)
2. Activates adenylate cyclase, catalysis cAMP, activating protein kinases
3. Causes excess secretion of Cl-, Na+ and Water due to excessive phosphorylation (activation) of ion channels by protein kinase A
4. Excess fluid and electrolytes lost in the lumen of small intestine
5. Diarrhoea and extreme dehydration
104
How is a Whooping Cough caused by Bordatella pertussis bacterium in 5 steps?
1. Inhibits Giα subunit (inhibitory)
2. Inactivation of inhibitory G-protein causes an increase in cAMP.
3. Causes too much intracellular cAMP
4. Leads to erosion of the respiratory epithelium and discharge of large quantities of mucus-containing fluid
5. Triggers coughing fits.
105
What are 3 examples of activating mutations of GPCRs and the effects?
1. Parathyroid Ca2+ sensor
>Hypoparathyroidism (failure to respond to serum Ca2+)
2. Rhodopsin receptor
>Night blindness
3. Thyroid hormone receptor
>Hyperthyroidism, thyroid cancer
106
What are 5 examples of Loss of Function mutations of GPCRs and the effects?
1. Cone cell opsin
>Colour blindness
2. Parathyroid Ca2+ sensor
>Hyperparathyroidism, (failure to respond to serum Ca2+)
3. Rhodopsin receptor
>Retinitis pigmentosa, retinal degeneration
4. Thyroid hormone receptor
>Hypothyroidism
5. Vasopressin receptor
>Nephrogenic diabetes insipidus; kidneys fail to resorb water
107
What subunit does 90% of uveal melanoma effect in GPCRs and how does cause the cancer to grow?
>Over 90% of uveal melanoma have mutations in Gq α subunit
>Leads to blocking of GTP hydrolysis so subunits always active causing permanent signal transmission causing uncontrolled growth (due IP3 increasing intracellular Ca2+ levels and DAG activating many protein kinase C promoting growth).
108
What trimeric G protein a) activates b) inhibits adenylyl cyclase
a) Activated by Gs alpha subunit
b) Inhibited by Gi alpha subunit
109
What trimeric G protein activates phospholipase Cβ and what is the effect of this?
Phospholipase Cβ is activated by Gq alpha subunit causing IP3 to be produced which causes release of Ca2+
110
How many isoforms of adenylyl cyclase are there?
10
111
What is adenylyl cyclase's relationship with a cell membrane?
Anchored on the intracellular side via 12 transmembrane domains (2 sets of 6 homologous proteins)
112
After stimulation of a Gs alpha subunit, what happens to an adenylyl cyclase enzyme to make it active?
C1a and C2a (carboxyl ends) of the enzyme bind making it in an active state.
113
What can imaging cAMP in a cell show us?
Can use the imaging to see that different parts of the cell are activated at different times dependent on the strength and length of that signal.
114
What are the 6 steps in cAMP second messenger system?
1) Ligand binds to receptor (extracellular N terminus) activating G protein
2) α subunit (of Gs protein) moves and binds to adenylate cyclase in the membrane
○ Occurs once GDP has been displaced by GTP in the alpha subunit.
3) This activated enzyme catalyses formation of a cAMP from ATP
4) The cAMP (2nd messenger) activates Protein kinase A
5) PKA phosphorylates/activates target protein
6) Initiates a response within the cell
115
What is an example response that cAMP second messenger system is used for and why?
Involved in fight or flight response as it is pretty quick for a GPCR mechanism.
116
How does the cAMP second messenger system work in the regulation of metabolism in liver and skeletal muscle cells in 6 steps?
1. Binding of single epinephrine molecule to β2 Adrenoceptor
2. Gs alpha displaces GDP for GTP and moves across the membrane
3. Activates adenylyl cyclase which converts ATP to cAMP
4. cAMP activates protein kinase A
5. Protein kinase A phosphorylates with the help of Ca2+ of protein kinase
6. Phosphorylated protein kinase converts glycogen to glucose using ATP, used as energy within muscles.
117
What are two differences between cGMP second messenger system and cAMP second messenger system?
1) Enzyme is guanylate cyclase which can be receptor bound or ‘free’ in the cytoplasm
>Not membrane bound adenylyl cyclase
2) Guanylate cyclase converts guanosine triphosphate (GTP) to 3’, 5’-cyclic guanosine monophosphate (cGMP)
>Instead of ATP -> cAMP by adenylyl cyclase.
118
What 3 factors determine the local concentration of 2nd messengers?
Determined by rate of production, rate of diffusion from site of production, rate of removal
119
What is the effect of a receptor taking up an increased concentration of Ca2+ on cAMP?
Phosphodiesterase is modulated by calcium calmodulin, so increased amounts causes increased conversion of cyclicAMP to 5'AMP so it cannot bind to proteins anymore like cAMP would.
120
What is the effect of cAMP when it is first produced?
cAMP levels are so high so bind to any protein with a binding site for cAMP
121
What takes place when a GPCR activates a IP3 and DAG second messenger system in three steps?
1. Activation of Gq alpha subunit, displaces GDP for GTP and moves across membrane.
2. Activates phospholipase Cβ which cleaves PIP2 ,in the membrane, into DAG (hydrophobic) and IP3 (hydrophilic/ water soluble)
3. IP3 moves through cytoplasm and activates an IP3 receptor/ Ca2+ channel on the intracellular membrane of endoplasmic reticulum, DAG stays in membrane and activates protein kinase C
122
What is a feedback mechanism which decreases DAG and IP3 signalling?
Lipid kinases add phosphate groups to DAG and IP3 to create PIP2 again
123
What creates diversity in the cAMP, IP3 and DAG second messenger systems?
Diversity and complexity in this pathway due to different isoforms of proteins (of phospholipase C and protein kinase C)
124
What is conserved in the different isoforms of a) Phospholipase C b) Protein Kinase C
a) PLC have X and Y catalytic domain conserved.
b) All PKC have conserved kinase domain
125
What are 3 examples of phospholipase Cβ differing in regulatory domains?
1) Have pH unit allowing binding to beta and gamma G-protein subunits
2) C2 regulatory domain allowing binding to lipids in membrane
3) Large carboxyl (CT) domain where alpha subunit binds.
126
What is similar about all Protein kinase C isoforms and what differs?
All PKCs are all serine/3NE kinases, they can bind to any protein with that binding site, but they are expressed in different tissues and activated by different molecules/ second messengers (most activated by DAG, only some by Ca2+)
127
What is PMA phorbol ester and its use?
PMA phorbol ester is an analogue of DAG used in research to activate PKCs
128
How does Protein Kinase C regulate itself from substrates binding?
Pseudosubstrate domain, in resting state it binds to the substrate binding site (mimic substrate) so no other substrate can bind there and be activated
129
What is the effect of DAG binding to protein kinase C?
DAG binding causes dissociation of intramolecular pseudosubstrate domain from active site, opening up substrate binding site for target proteins, once activated PKCs can provide either positive or negative feedback in signalling pathway
130
How does binding of PKC to phospholipase Cβ cause negative feedback?
Phosphorylation of PLCβ caused by activation of protein kinase C by DAG provides negative feedback for GPCR signalling, makes the signalling ‘transient’ (limited duration of time) and phosphorylates GPCRs causing depolarization.
131
What is the positive feedback that can be caused from PKC binding to phospholipase Cβ?
Phosphorylation of PLCβ caused by activation of protein kinase C by DAG can cause negative feedback onto GPCR receptors where they are phosphorylated and desensitization
132
What are 5 examples of processes regulated by the second messenger Ca2+?
1. Synaptic transmission
2. Hormone secretion & synthesis in some cases
3. Fertilization
4. Muscle contraction
5. Cytokinesis
133
What concentration of Ca2+ does the endoplasmic reticulum contain, how does this compare to intra and extra cellular concentrations?
>Endoplasmic reticulum contains 400um Ca2+ conc which is higher than intracellular conc (100nm) but lower than extra cellular conc (2.5mM)
>So is a Ca2+ store.
134
How is the conc of Ca2+ in a resting cell kept low and why is this important?
>by ATP-driven Ca2+ pumps
>Important for Ca2+ to be able to act as a 2nd messenger
135
What are the 2 regulatory pathways of increasing intracellular Ca2+?
1) By IP3 binding to ligand gated Ca2+ channels on the ER, causing them to open and due to high conc of Ca2+ stores, Ca2+ rushes into the cytoplasm of the cell.
2) Activation of IP3 Ca2+ channels on ER (amplifies signal of IP3) activates STIM which binds to Ca channels called ORAI on the plasma membrane of cell, allows Ca2+ to flow into the cell from extra cellular fluid.
> But ORAI activation also cause activation of calcium pumps re-fill ER with Ca2+ as they are needed for protein production.
136
What is the effect of a loss of function mutation in ORAI 1?
Loss of function mutation in Orai1 causes severe combined immunodeficiency (SCID) as the channel plays a key role in activation of T-lymphocytes. (Maybe just say, Less C2+ influx from extracellular fluid when Gq pathway is activated, and the ER stores of Ca2+ aren't re-filled).
137
What is a method used to study Ca2+ second messenger signalling to allow in vivo recording of calcium signalling associated with different behaviours?
Tag with GFP via genetically engineered animals.
138
What are two outcomes of overstimulation of GPCRs?
1. Tachyphylaxis - continually exposed to drugs or medication e.g. LSD or salbutamol
>Can build up resistance to it, so takes more of the drug to cause effect
2. Disease e.g. uncontrolled growth in cancer
>If GPCRs are constantly stimulated can cause uncontrolled growth.
139
What 2 GPCR mechanisms regulate desensitization?
2 mechanisms that occur early on in pathways to regulate this desensitization - GRK (receptor kinases) and β-arrestin
140
How do 1) GRK and 2) β-arrestin regulate desensitization of GPCRs?
1) GRK binds to binding site for G-protein, which stops G-protein from binding so signalling never occurs
2) β-arrestin phosphorylates the receptor acting as a signal for proteosomes to degraded or recycle the receptor
141
What is the effect of mutational modifications of receptor kinase and an example?
>Mutational modification of receptor kinases causing the gene not to be expressed
>e.g. Can lead to prolonged proton response and rod apoptosis in Rhodopsin receptor
142
What are the 4 ways GCPR signalling is switched off?
1. Agonist dissociating from receptor
2. GTPase activity of Gαs causes GDP to be present instead of GTP
3. cAMP breakdown by phosphodiesterase
4. Dephosphorylation of enzymes
