Cell Phys Flashcards
What are ABC transporters?
- Transport system superfamily
- Largest protein family so far with 1-3% of genomes coding for their subunits
What is the general function of ABC transporters?
Unidirectional importing and exporting proteins against their chemical gradients
Eukaryotes- exporting
Describe the molecular structure of ABC transporters
Characterised by:
- Two nucleotide binding domains (NBDs)
- Two transmembrane domains (TMDs)
Also has:
- Phosphate- binding loop (P-loop)
- Short signature sequence (LSGGQ)
What have studies observed about the gating of ABC transporters?
Balarkrishnan et al. - drug efflux pump LmrA has been shown to be reversible in certain conditions.
This suggests that the membrane domain has ‘turnstile’ like gates rather than barriers.
What are the problems with the ATP switch model?
There is the general agreement that these steps must occur at some point but less understanding of the exact order.
List examples of human ABC transporters
P-glycoprotein (p-gp) MRP1 ABCA1 SUR1 (ABCC8) CFTR
Describe the functions of p-glycoprotein
- Transports neutral and cationic hydrophobic compounds
- Transports xenobiotics out of cells
- Exploited by tumour cells
- Expressed in filter organs (e.g. liver, kidney, intestine…)
What does p-gp have to do with multi-drug resistance?
Expressed at high levels in tumours -> transports anticancer drugs out of cancer cells
How does p-gp know what the cytotoxic level of anti-cancer drug is?
1- Already a small population of p-gp which survives and replicates via natural selection during cancer treatment
2- All cells have small concentration of p-gp but the instability of the cell cycle means that the tumour cell can up regulate p-gp expression
What is the role of MRP1?
Transports anionic compounds
What is MRP1 named a ‘multi-drug resistance associated protein’?
It’s expressed in most tissues (and therefore present in cancers).
This means that like p-gp, it can export anticancer drugs out of the cell.
What is the role of ABCA1?
Cholesterol efflux from:
- Macrophages
- Placenta
- Liver
- Lungs
- Adrenals
Describe the consequences of ABCA1 mutation
Tangier disease
Familial HDL deficiency, characterised by:
- Low HDL levels
- Lipid dense macrophage deposits in tissues
- Atherosclerosis and associated diseases
How does ABCA1 mutation cause familial HDL deficiency?
- Usually, app A can strip lipids from the membrane via the ABCA1 receptor
- ApoA can then get loaded, becoming a mature HDL particle which goes to the liver to get discarded.
- Stopping the ABCA1 receptor stops cholesterol and lipid from leaving the cell so it accumulated.
- Cells can get deposited anywhere - plaque formation more likely.
What is the role of SUR1?
- Interaction with KATP channels
- Insulin secretion
Describe the structure of the SUR1 receptor
- 4 SUR1 subunits
- Pore formed by 4 Kir6.2 subunits which potassium can move through when unblocked
How can the SUR1 channel be inhibited?
- Sulfonylureas (e.g. metformin) to NBD
- ATP or ADP to the Kir6.2 subunit
How can the SUR1 channel be stimulated?
MgATP and MgADP binding to NBD1 and NBD2
How do we know how KATP channels behave in insulin secretion?
- Calcium channels are always open whenever there is depolarisation
- Where calcium channels are open, potassium channels must be closed and vice versa
Describe medical problems which affect SUR1
Hyperinsulinism – beta islet cell membranes are always depolarised (due to MgADP insensitivity) -> increased insulin release-> low glucose
Neonatal diabetes – beta islet cell is always hyperpolarised (due to ATP insensitivity) so insulin is never released -> high glucose
What is the function of the ABCC7 transporter?
CTFR- transport of chloride ions out of cells
How is the gating of the CFTR protein regulated?
- Protein kinase A (PKA)- mediated phosphorylation
- ATP interaction (Muallem 2009)
- Nucleotide content of nucleotide binding domains (Wilkens 2015)
What is important for CFTR activation?
CFTR needs to be phosphorylated by PKA in the presence of ATP
What is the difference between SUR1 and CFTR?
SUR1 regulates electrical conductance, CFTR is just a chloride ion channel
What kinds of mutations occur to the CFTR protein?
Delta- alpha508 - channel partially active and stuck in endoplasmic reticulum so it can’t transport Cl- ions
How does the CFTR protein close?
Nucleotide of the dimer dissociates (likely due to instability) and the channel closes.
Outline the stages of CFTR transport with reference to the ATP switch model
- ATP binds, channel opens so Cl- can travel through; inward movement of binding site
- PKA phosphorylates, triggering outward movement of binding site
- Complex becomes unstable
- ADP association
What are the normal intracellular and extracellular concentrations of calcium?
Intracellular- 100nM
Extracellular- 2mM
Outline the underlying mechanism behind hyperalgesia
TRPV1 is under the influence of many messengers
Increased receptor function -> hyperalgesia
What is the difference between allodynia and hyperalgesia?
Allodynia - touch is associated with pain, provoking nociceptors -> neuroma formation
Hyperalgesia- pain stimulus is present but individuals have higher sensitivity
Outline the rationale behind functional cloning
- Look at which receptor responds to capsaicin
- Take the whole RNA for that receptor
- Make a library
- Express that library in a cell you know doesn’t normally express it
- If the cell that normally doesn’t perform the function suddenly does after you add the library, you know it has to be down to the RNA that was added.
What are the response properties of nociceptors?
Adaptation- for a constant stimulus, action potentials slowly decrease
Fatigue- after adaptation to a constant stimulus, they need time to recover
Sensitisation- after injury, this is specific to heat and mechanical stimuli
What are the stages of encoding and processing noxious stimuli?
Transduction - detecting noxious stimuli
Conduction - transmission of information along the nerve fibres
Transmission - at the first pain synapse in the spinal cord
How is pain distinct from other sensory stimuli?
- Receptor sensitivity
- Pain only becomes pain once nociceptors have interacted with the limbic system
Where does specialised detection of pain signals take place?
Upper lamina in the substantia gelatinosa
What are the constituents of ABC transporters in eukaryotes?
Single polypeptide with all four functional units
Some units assembled with ‘half’ transporters; can be homodimeric or heterodimeric.
What is the nucleotide binding domain otherwise referred to as?
ATP-binding cassette
i.e. the hallmark of the ABC transporter family
What is responsible for the identification of nucleotide binding domains of ABC transporters?
Signature sequence LSGGQ
Describe the properties of transmembrane domains
- 6-10 transmembrane alpha-helices (most exporters have 6)
- Since some are dimers, that leads to a total of 12-20 respectively.
What is the difference between ‘inward’ and ‘outward’ facing transporters
Inward - pore accessible from cytoplasm
Outward - pore accessible extracellularly
Why has p-glycoprotein been described as having ‘polyspecificity’ towards its transport substrates?
It has several overlapping drug-binding sites.
How do ABC transporters move substrate with respect to their chemical gradients?
With few exceptions, ABC transporters pump them against their gradient.
Give an example of a eukaryotic ABC importer
Vitamin B12 uptake
With reference to the ATP switch model, describe how ABC exporters work
- Substrate binding to the transmembrane domain (TMDs)
- ATP molecules bind to the nucleotide binding domains (NBDs)
- NBD dimerises, TMD change in conformation (inward -> outward)
- ATP hydrolysis; NBD dissociation, phosphate, ADP and substrate release
Cycle then resets transporter to ground state
What is the proposed mechanism of ATP hydrolysis in the ATP switch model?
Base catalysis by glutamate residue at the end of a phosphate binding loop.
What is the ABCC1 transporter more commonly known as?
MRP1
What is the ABCB1 transporter more commonly known as?
P-gp
List the ABC transporters involved detoxification
ABCB1
ABCC1
ABCG2
What does ‘degenerate’ mean with respect to ABC transporters
- SUR1 and CFTR both have one of these as their nucleotide binding domain.
- They can still bind ATP but it isn’t hydrolysed as efficiently.
What is the ABCC8/9 transporter more commonly known as?
SUR
List the different types of carriers/ transporters
Uniporters
Cotransporters
Antiporters
What is the difference between carriers and channels?
Only carriers carry out active transport
Carrier- alternates between two conformations so solute binging site alternates in accessibility
Channel - from water-filled pore across bilayer that’s ion specific
Compare symporters and antiporters
Both examples of coupled transport
Symporters- transport molecules in same direction
Antiporters- transport molecules in opposite directions
Why are carriers useful for diffusion?
- Transport rate is higher
- Saturable process
- Kinetics are similar to enzyme catalysis
What is the difference between ion channels and single aqueous pores?
Ion channels:
- have a selectivity filter
- aren’t always open- they’re gated
What are the distributions of intracellular and extracellular sodium and potassium?
Sodium
Intracellular - 10mM
Extracellular - 140mM
Potassium
Intracellular - 145mM
Extracellular - 4mM
What is the function of specialised channels and pumps?
Specialised channels conduct ions down their electrochemical gradients
Specialised pumps maintain ion gradients by pumping ions against their gradient
How do you determine how substrate flux (movement) is mediated?
Generally speaking, channels have higher conductance levels than pumps though the distinction is not foolproof
What is the difference between ion channels and ion exchange pumps?
Ion channels only need one gate which can be open/ closed at any one time
Ion exchange pumps need two gates which are never both open at the same time
How do glutamate transporters behave?
Carrier/exchanger for glutamate, coupled with protons, sodium and potassium ions
Channel for chloride ions
Describe the structure of a bacterial glutamate transporter
Trimer
Structural evidence suggests that each subunit binds its amino acid substrate and acts as a transporter/ chloride channel independent of the other monomers.
Why are nociceptors ‘psuedounipolar’?
They don’t have dendrites
Where are the majority of nociceptors found?
Dorsal root ganglion (peripheral nervous system)
How are noxious stimuli detected?
Free nerve endings detect tissue damage and noxious signals via:
- Chemicals
- Temperature extremes
- Mechanical insults
Describe the cellular diversity of nociceptors
Alpha beta - low threshold mechanoreceptor (fastest)
Alpha delta - high threshold; cold, pressure, chemical
C - polymodal; temperature, chemical and pressure
What is ‘nocifensive’ behaviour?
A sign of pain, affective response (e.g. jumping, withdrawal) and conditioned motor response (e.g. avoidance, escape, aggressiveness)
How can the qualities and temporal features of pain be described?
First - stabbing/ pricking (acute)
Second - burning/ throbbing/ cramping/ aching; more affective to patients (chronic)
Outline the neural pathway for nociception
First pain synapse -> dorsal horn -> central nervous system (including limbic system)
Where do alpha-beta myelinated fibres synapse in the spinal cord?
PKC-gamma+ neurons
Lamina V
Where do peptidergic C fibres synapse in the spinal cord?
Lamina I
Outer lamina II
Where do alpha-delta myelinated fibres synapse in the spinal cord?
Outer lamina II
Lamina V
Where do non-peptidergic C fibres synapse in the spinal cord?
Inner lamina II
What is the general response of nociceptors to hot and cold stimuli?
As the temperature (hot temperatures increase) becomes more extreme, action potentials increase.
As soon as temperature reaches threshold, the number of action potentials are massively increased
What are nociceptors?
Axons, cell bodies and central terminals of nociceptive dorsal root ganglion neurons
Which nociceptors are the free endings generally associated with?
Unmyelinated C fibres
Thinly myelinated alpha-delta axons
What is neurochemically understood for nociception?
- Neurofillaments in alpha- beta fibres
- IB4 (sugar molecule) positivity in alpha-delta
- Neuropeptides (peptidergic C fibres) express CGRP, an important pain indicator
How are noxious stimuli transduced with respect to action potentials?
As stimulus intensity increases, so does membrane potential until threshold is crossed and an action potential generated.
What quality of nociception makes chronic pain a problem?
Nociception doesn’t really adapt but do fatigue
This means that for a patient suffering from chronic pain, it would come and go but remain consistent (with increased action potential firing before fatigue)
How are different temperatures detected and integrated in nociceptive signalling?
- Different transient receptor potential (TRP) channel combinations
- Temperature detection by subset of DRG neurons
What sensory neurons are responsible for detecting cold?
M8
A1
What sensory neurons are responsible for detecting heat?
A1
V1 - 40-50 degrees
V2- 60 degrees
What is the most famous gene associated with mechanoreception?
Piezo2
What is spontaneous pain?
Pain that is not evoked by a stimulus
What is the role of substance p with respect to pain?
Effector, released by DRG neurons and responsible for:
- Vasodilation
- Initiation of most cytokine expression
- Amplifies/ excites most cellular processes
What are chemical analogues for noxious temperature stimuli?
Capsacin - heat
Menthol - cold
List examples of investigated candidates for the sensation of mechanical pain in mammals
- OSM-9 (in C.elegans)
- nompC
- Nanchung
- Painless (Drosophilia)
- TRPA1 (mammalian hearing)
- TRPP2 (mechanoreception in epithelial cells)
How might peripheral sensitisation lead to pathological pain?
- Augmented TRP channels -> sensitisation
- Modulation of sodium and potassium channels -> altered nociceptor excitability
What are reactive oxygen species (ROS)?
Any oxygen species that has the strong ability to accept electrons, reducing itself. Effective but non-selective killers They include: - Oxygen ions - Free radicals - Peroxides
What is the difference between the outcomes for high and low oxidative stress?
High:
Increased expression of ‘atherogenic’ genes resulting in inflammation and vascular dysfunction
Low:
Increased expression of ‘atheroprotective’ genes - anti-inflammatory and protective for vasculature
What is Nrf2?
NF-E2 related factor 2
Member of the cap-n-collar family; master regulator of cell responses against environmental stresses
What is the role of Nrf2?
Induces expression of phase II detoxification enzymes and antioxidants
List examples of ROS generated diseases
- Diabetes
- Pre-eclampsia
- Atherosclerosis
- Uraemia
Describe KEAP 1 and its function
Kelch-like ECH-associated protein; subunit of ubiquitin ligase
Regulator of Nrf2
How does KEAP 1 sense stress?
Its cysteine residues can detect different stress stimuli
How does the Nrf2-KEAP 1 pathway change with redox?
- Oxidation of KEAP 1 cysteine residues
- Conformational change in Nrf2/ KEAP 1 complexes
- Nrf2 forms heterodimers with May binds to antioxidant responsive element in promoter region of target genes
- Expression results in production of proteins to help manage stress (e.g. HO-1)
What are possible problems with excessive Nrf2?
- Oncogenesis and cancer cell resistance
- Overproduction of reduced glutathione and NADPH
- Failure of corrupt differentiation in some cells
What are the consequences of too little Nrf2?
- Loss of cytoprotection
- Diminished antioxidant capacity
- Lowered beta-oxidation of fatty acids
- Foetal pre-disposition to vascular disease
What is oxidative stress?
Protein oxidation that's poorly reversible by anti-oxidant systems; produced by the burden of ROS exceeding its antioxidant capacity Can be caused by exposure to: - UV radiation - Environmental pollutants - Cigarette smoke
What are antioxidants?
Small molecules that possess redox- active properties, capable of scavenging ROS/ reactive nitrogen species.
What is Nrf2 modulated by?
PKC
MAPKs
What is the role of NQO1?
Phase II detoxifying enzyme stimulated by HNE
How does Nrf2 play a role in pre-eclampsia and the offspring’s predisposition to vascular disease?
- Less Nrf2 in foetal endothelial cells- less nuclear translocation and ARE binding in stress
- Reduced HO-1 expression
- Foetal cells more sensitive to HNE induced DNA fragmentation
- Impaired antioxidant gene expression
How is Nrf2 affected in gestational diabetes?
- Diminished translocation to the nucleus -> less dimerisation with Maf
- Less HO-1 induction via HNE in aortic smooth muscle
- Compromised ARE genes from sustained activation via oxidative stress
How is Nrf2 affected in ageing?
- Decreased nuclear level
- Down-regulation of pathways like glutathione synthesis
How is calcium usually found in cells?
Bound to calsequestrin in ER
Free/ bound to calmodulin in cytoplasm
What is a general rule for the speed of different active transporters?
Primary - slow
Secondary - fast
How can Nrf2 be degraded without KEAP1?
GSK-3beta phosphorylation via an adaptor protein (beta - TrCP)
What is maf?
Antioxidant response element
How does Nrf2 play a role in acute inflammation?
Regulates prostaglandin production through transcriptional regulation of: - Peroxiredoxins 1 and 6
- Liopocalin-type prostaglandin D synthase
Why is Nrf2 a double edged sword?
Activation of some of the genes it binds to could increase resistance to chemotherapy in cancer (e.g. MRP1, p-gp, ABCG2)
How does calcium signalling link to ROS?
- Increased mitochondrial Ca2+ boosts ATP production
- Increased ATP production means more oxygen is being reduced to water during oxidative phosphorylation
- More reduction means more leakage of free electrons
- More free electrons means more formation of superoxides
Where are ROS generated by living cells?
- NADPH oxidases
- Xanthine oxidase
- Lipoxygenase
- Diaphorase
- Mitochondrial electron transport chain (ETC)
- Uncoupled endothelial nitric oxide synthase (eNOS)
List examples of ROS that can cause protein modification
- Peroxynitrite
- Superoxide
- Hydrogen peroxide
- Hydroxyl radicals
How does 4-hydroxynonenyl (4-HNE) modify proteins?
Michael Adduct formation; thought to lead to abnormal function
How does peroxynitrite modify proteins?
- S-nitrosylation of cysteine
- Nitration of serine, threonine, tyrosine
These all lead to modified function (unclear of whether this is bad)
How do superoxide and hydrogen peroxide modify proteins?
Both cause reversible cysteine sulfenylation.
Problem: the more electrons you add, the more difficult it is to reverse the process.
How is reversal of protein modification possible?
REDOX buffers (primarily glutathione) which are catalysed by reductases
Give examples of ROS sensitive signalling in vascular smooth muscle
- Tyrosine phosphatase inhibition (cysteine are oxidised)
- Kinases (direct oxidation or oxidation of GPCRs)
- GEFs (indirect activation)
- Src activation
How does ROS affect protein kinase C activity?
Depends on dose.
Low doses - stimulatory
High doses - inhibitory
Why is the electron transport chain considered to be a hypoxia sensor?
- Enhances leak of electrons, increasing superoxide production
- Increased superoxide production -> increased ROS -> increased contraction
- Block complexes, prevent superoxide generation, reduce ROS etc.
What are the effects of rotenone and succinate?
Rotenone - blocks ROS production via complex I
Succinate - overrides effect of rotenone
How does ROS cause contraction?
Increasing calcium influx into the cell -> activation of MLCK -> phosphorylation of MLC
Or MLCP inhibition:
- Src (via PKC) activates CPI-17
- Rho A (via ROCK) activates MYPT
What are the effects of exogenous superoxide on the mesenteric artery?
Mixed:
- Low doses - enhanced contraction
- High doses - inhibited contraction
What is endothelium-derived hyperpolarising factor (EDHF)?
Any factor that causes hyperpolarisation and therefore relaxation of underlying smooth muscle
What is the difference between coupled and uncoupled eNOS?
- Coupled-> vasodilation (product is nitric oxide)
- Uncoupled (via BH4 oxidation) -> vasoconstriction (product is hydrogen peroxide which is ROS and EDHF)
Why is hydrogen peroxide more likely to be pro-relaxant?
- It doesn’t activate the rho-kinase pathway or scavenge NO like peroxinitite
- Likely just diffuses into vascular smooth muscle where it can activate K+ channels
Why are ROS considered pro-proliferative?
They activate ERK and TRK which are both pro-proliferative
