Intracellular Signalling Flashcards
Name some of calcium’s roles in signalling (8)
- Synaptic transmission
- Neuronal excitability
- Signal amplification - 2nd messenger
- Synaptic plasticity
- Regulation of gene expression - (in neuron) regulates TF’s eg Kreb
- Axonal growth - guidance + branching - steer dev axons to target cells + formation
- Neuronal survival
- Regulation of neuronal cytoskeleton
EF hand proteins background (4)
also known as calcium binding proteins
-120 families of EF hand proteins
- EHelix-loop-Fhelix
- present in wide variety: bacteria to humans
- various roles eg muscle contraction, enzyme activation, gene expression reg, neurotransmitter release
Name + explain an EF hand protein (3)
Eg : calmodulin and parvalbumin
calmodulin:
- found in almost all eukaryotic cells
- regulates the activity of numerous enzymes + other proteins by binding to ca2+ ions and transmitting ca2+ signals to downstream targets
how do EF hand proteins work? (3)
1) bind to ca2+
2) confirmational changes to protein
3) = allows them to sense [ca]i changes = sensors
How do we measure calcium + eg? (4)
using chemical or protein based fluorescent indicators - (has EGTA bound to it)
eg. Indo-1
- emits fluorescence once bound to 2 calcium ions
- usually emits light around 350-380 nm
- shorter + longer wavelengths ratio analysed = monitor changes in [ca]i
Calcium enters through… (4)
- Voltage operated channels (VOC)
- Ligand operated channels (LOC) - + other non-selective channels: Ach r’s + ATP r’s = ALLOW INFLUX OF CA2+ INTO CELL
- Store-operated channels (SOC) - empties ER ca2+ store
- Transient receptor potential (TRP)
How does the removal of excess calcium take place? (3 + 3)
- Plasma membrane Ca2+–ATPase (PMCA) :
- form phosphorylated intermediates during x cycle + calmodulin binds to c terminal at certain splice variants
- ca2+ + calmodulin = confirmational changes
- Na+-Ca2+ exchanger (NCX):
- at rest: 3na+ for 1ca2+
- bidirectional
- voltage dependent = reverse their exchange na+ during AP’s = NA out of cell and Ca into the cell
Endoplasmic reticulum background (2)
- Primary intercellular calcium store - spanning large distances = can move ca2+ in and out of cells w/o detrimental effects eg local control as opposed to global - accumulation
3 main compartments:
* Smooth
* Rough
* Nuclear envelope
Calcium retrieval into the ER (4)
- [Ca2+] 100 to 800 mM (microM in cytoplasm)
- SERCA pump facilitates transfer - energy intensive pump requires the hydrolysation of ATP
- 2 Ca2+ ions transferred for each ATP –> moves across the gradient
SERCA 1 + 2: muscle
SERCA 3: other tissues eg brain
Release of calcium from ER to cytoplasm (4)
From either r:
-Ryanodine (RyR) receptor
- inositol(1,4,5)-triphosphate receptor (IP3R)
==> Both release channels from tetramers of identical subunits + have long Nterminal regions = binding sites for ATP, Ca + proteins (eg calmodulin)
They’re activated by:
-IP3
- calcium
-cyclic ADP ribose (cADPR)
What’s the role of the mitochondria on Ca2+ dynamics? (3)
- Can store vast quantities of calcium - but has to be regulated to avoid overproduction of ROS ( +inhibition of ATP + mito depol)
- positioned in cytoplasm near areas of high [Ca2+]
- Detect high [Ca2+] and produce energy
How does the mitochondria detect high [Ca2+] and produce energy? (7)
close relationship w/ER - bound by tethering proteins eg microtubules anchoring them together
1) ca release from ER in localised system
2) VDAnionC in atrial membrane supplies ca2+ to transporters in inner mito membrane = accumulation of ca2+ within mito.
3) elevated ca2+ = activates rate limiting step in TCA cycle = increased oxidative phosphorylation + promotes ATP synthesis
4) ADP transported from mito via adenine nucleotide transporter in exchange of ATP
5) Ca2+ exits mito via NA+/Ca2+ or Ca2+/H+ echanges = sequestered in ER by SERCA
6) necrosis occurs when high ca2+ w/ oxidative stress
7) = opening mito transit pores (MTP) = loss of ATP
What is a drug - definition? (2)
A chemical substance of known structure, other than a nutrient or an essential dietary ingredient which, when administered to a living organism, produces a biological effect.
With some exceptions, drugs act on target proteins, namely:
* receptors
* enzymes
* carriers
* ion channels.
What is a receptor - definition? (1)
Protein molecules whose function is to recognise and respond to endogenous
chemical signals
What is the two-state model of receptor activation? (5)
(image)
Resting <-> activated r* -> response
- no ligand: high to low (skewed to resting)
- ligand/endo r: low to high prob (high affinity for activated state = more likely to produce response)
- Agonist: affinity R*>R
- Antagonist: affinity R=R*, reduced availability of binding sites for other ligands
Drug def (1)
Chemical applied to a physiological system that affects its function in a specific way
Ligand def (1)
Any molecule or atom which binds reversibly to a protein
Agonist def + types (3)
Drugs which ‘activate’ receptors
-full: = activated until r saturation/ max effect
- partial: partially elicits a partial response
Antagonist + types (1 + 4)
A drug that binds to the receptor without causing activation
- neutral: binds but no effect
- inverse: binds but inverses effect
-Reversible: compete w/ agonist binding typically at the same site. Binds reversibly - Irreversible (covalent): Binds irreversibly to receptor. May change the conformation of the receptor to reduce ability of agonist to bind
what 2 factors are often considered when looking at conc + effect graphs? (2)
Emax
EC50
What’s Emax? (1)
The maximal response
What’s EC50? (2)
The concentration of a drug that gives half-maximal response
- gives drug’s potency
Orthosteric site def (1)
The primary ligand binding site of a receptor
Allosteric site def (1)
A site distinct from the endogenous ligand
Allosteric modulators
–Allosteric modulators impact receptor function by binding at a site distinct from the endogenous ligand
How does allosteric modulation impact dose response curves? (affinity vs efficacy) (4)
image
- positive AFF mod: moves left
- negative AFF mod: moves right
- positive EFF mod: moves up
- negative EFF mod: moves down
What are G proteins? (4)
- Protein family that act as molecular
switches inside a cell - Heterodimeric complex made up of a, b and g subunits
- Activated by GCPRs (metabotropic r’s)
- Many neurotransmitters react w/ both ligand gated ion + GPCRs
What’s the heterotrimeric G-protein? (3)
αβγ subunits:
alpha
beta
gamma
attached to GDP
How do GPCRs flip on a G-protein switch? - MoA (5)
1) Agonist (eg LH) binds to the r = causing conformational change in the GPCR
2) Change in r conformation activates heterodimeric G protein (αβγ) : inactive GDP bound –> active GTP bound
3a) GTP bound to α dissociates from βγ
3b) The activated G protein the activates an effector enzyme (e.g. adenylyl cyclase)
4) Effector generates an intracellular 2nd messenger (e.g cAMP)
How do you flip off the G-proteins switch (2)
5) GTPase activity: α controls GTPA’s activity => binding of subunit to target = catalyses GTP -> GDP (inactivation of α)
6) α reforms w/βγ = αβγ but remains inactive
Structure of abg protein (4)
- crystal structure
- 7 TM domain
- G Protein binds to 3rd cytoplasmic loop
- r smaller than G protein
Egs of GCPRs (5)
Examples include:
* Adrenergic
* Dopaminergic
* Opioid
* Glutamatergic
* GABAergic
What do all these GPCRs do? (4)
Functions include:
* Light detection
* Odorant detection
* Hormone detection
* Neurotransmission
What are the 3 main families / classes of GPCRs + how are they classified? (5)
Heterotrimeric G-proteins (αβγ):
Family A
Family B
Family C
- Genetic: high sequence homology within each family(the genetic sequence of the proteins within each family is very similar)
- Structural: Each family has a distinct location of agonist binding domain and length of extracellular N-terminus
Family A egs (6)
- Adrenergic
- Rhodopsin
- Opioids
- Odorant
- Adenosine
- Cytokines
Family B egs (3)
Receptors for peptide Hormones
* e.g. Glucagon,
* Growth hormone
* Parathyroid hormone
Family C egs (4)
- Metabotropic glutamate receptor (mGluR)
- GABAB receptor
- Pheromones
- Taste receptor
How do G-proteins transduce and amplify signals? (4)
1) 1 ligand binds to 1 r
2) r can signal to eg 4 bound G proteins
3) G proteins go + activate multiple effector enzymes (2 amplifications of the response)
4) effector enzymes further amplified by acting on many diff targets
How does each kind of receptor produce a distinct pattern of cellular effects - eg adrenaline? (2)
- Receptors activate different types of G-proteins
- There is molecular variation in α-subunit of the G-protein
Not all G proteins are the same - explain (3)
In humans:
16 Gα
5 Gβ
12Gg
-> These can combine to form different heterotrimeric G proteins
- There are 4 Ga subunit families that are classified by sequence, effector targets and GPCRs
- Gαi/o
- Gαs
- Gαq
- Gα12/13
2nd messengers egs (3)
- Membrane associated, water insoluble
e.g. diacylglycerol (DAG) - Cytosolic location, water-soluble
e.g. cAMP, cGMP and Ca2+ - Gases
e.g. nitric oxide
Properties of Metabotropic Glutamate Receptors (6)
Depending on subtype can be pre- and/or postsynaptically localised
Generally play a modulatory role in synaptic transmission
Postsynaptic group I mGlu receptors mediate slow depolarization (EPSP)
Presynaptic group II and III mGlu receptors decrease neurotransmitter release
Involved in the modulation of signalling through K+ and Ca2+– control excitability of
neurones
Metabotropic glutamate receptors were desirable targets for drug
discovery.
Metabotropic Glutamate Receptor Family (mGlu1-8) subtypes (3)
Group I: mGlu1 & mGlu5
Group II: mGlu2 & mGlu3
Group III: mGlu4, mGlu6, mGlu7, mGlu8
R’s placed into 3 groups according to AA sequence
homology, signal transduction mechanism and agonist pharmacology
Group selective
Agonists for mGluR (3)
I: DHPG
II: LY404039
III: L-AP4
Group I: Gq (4)
ca2+ dependent
1) glut binds to 1/2/5 r = dissociation of alpha subunit (GDP-> GTP)
2) GTP binds to PLC =
breakdown of PIP2 into IP3 + DAG
3) IP3 acts on IP3r in ER
DAG activates phosphokinase C
= inc. [Ca2+]I
Groups II + III: Gi/Go (4)
cAMP dependent
Activation of Gi/o coupled receptors:
bind - dissociation of alpha subunit (GDP-> GTP)
1) Inhibits cAMP production
2) Activates GIRK K+ channels
3) Inhibits voltage-sensitive Ca2+
channels
mGlu Receptor structure (4)
- Bi-lobed N-terminal extracellular domain - glutamate binding site (orthosteric site)
- Cysteine rich domain = maintains
tertiary structure - 7-TMD in
common with other GPCR families (allosteric
modulators bind at sites in the TMD) - 2nd intracellular loop involved in G-protein
coupling and in determining transduction
mechanism
X-ray crystallography of the ligand binding region reveals a homodimeric structure for mGlu1(6)
The ligand binding region (LBR) of mGlu1 has been expressed in
soluble form
X-ray crystallographic analysis showed mGlu1= homodimeric
The 2 protomers connected by a disulphide bridge b/w cysteine residues present on both LB1s of the LBRs
Bilobed structures (LB1 + LB2) of each protomer are flexible +can form open or closed conformations
X-ray structure showed Glu bound to both protomers one with
lobes closed and one with lobes open – corresponds to an activated state
Research suggests that lobes should be closed in both protomers for fully activated state.
Describe the constitutive activation of group I mGluRs + eg (2)
Spontaneous lobe closure in the absence of the agonist can lead to mGlu receptor activation (constitutive activity) + basal activity of the receptor
e.g. basal levels of phosphoinositide hydrolysis – blocked by inverse agonists
What does glutamate binding do to mGluRs? (1)
Glutamate binding stabilises the activated statewhile orthosteric antagonists bind to the resting state to prevent the lobe closure that would lead to receptor activation
Explain how MPEP is a Negative Allosteric Modulator of mGlu5 (3)
MPEP = Negative Allosteric Modulators
(NAMs)
binds to TM region AA residues ii + iii in mGlu5 to inhibit the glutamate-stimulated rise in intracellular [Ca2+]
Increasing concentrations of MPEP decrease the maximum response of a glutamate conc response curve on rat mGlu5
Positive Allosteric Modulators (PAMs) e.g.
Ro 01-6128 (3)
binds to AA residues in TMIII and TMV
Concentration-response curves for the effect of (S)-DHPG on mGlu1 are shifted to the left in the presence of Ro 01-6128
A 5-fold decrease in EC50 value and a 1.2-fold increase in the maximum response was observed in the presence of Ro 01-6128 i.e. change in efficacy of orthosteric agonist DHPG
Why is there a Slow EPSP from mGlu1 Receptor compared to ionotrophic r’s? (3)
because they’re not largely driven by an ion chance but the release of ca2+ from stores
= longer time to see ESPC (slower + more mediated = cant see)
==>biphasic distribution seen:
- fast depol by AMPAr + glut r
- much slower depol by mGlutr
