Membranes Flashcards
What are the three main functions of the membrane?
- Nerve transmission (action potentials). 2. Membrane trafficking. 3. Signalling.
What can biochemical systems be described as?
Modular.
Why is it thought at biochemical systems are modular?
As this would have been the simplest way for it to evolve.
Why would membranes first have arose?
As a barrier.
What was suspected to be in the membranes of early organisms?
Channels.
What evolved in membranes after channels?
Pumps (the ability to move things with a concentration gradient.)
What is an example of something regulated via pumps in membranes?
Glucose intake.
What can membranes convert a membrane potential gradient into?
Energy- such as with the ATP pumps.
Cell recognition is different in prokaryotes and eukaryotes. Why is thought to be the case?
The last universal common ancestor didn’t do this- thought to have evolved later as a late evolutionary adaptation.
Is signalling universal?
No, only similar.
What type of cell does compartmentalisation occur in?
Eukaryotes.
What is the average dimensions of E.coli?
1um by 2um.
How much bigger is a mammalian eukaryotic cell compared to a E.coli cell?
1000x bigger (10X bigger along each dimension).
Fibroblasts in the skin layer are not as thick as the epithelial cells. How much times wider are they?
4.
How much longer is a nerve axon compared to an epithelial cell?
500,000 times longer.
Why is compartmentalisation vital in eukaryotes?
As the cell is much bigger and diffusion will not be fats enough.
How much plasma membrane is in in a eukaryotic cell?
700 um2.
How much internal membranes are in the prokaryotic cell?
7000 um2.
What can you make membranes spontaneously aggregate into in a lab?
Lipid bilayer, liposome, vesicle.
Are vesicles or liposomes spherical?
Liposomes.
Are vesicles or liposomes made of layers?
Vesicles.
What direction of movement is fast in a membrane?
Lateral.
In cells what will membranes spontaneously aggregate into?
Just bilayers.
Lipid anchor proteins are solvable proteins with what added to them?
Hydrophobic tail.
Lipid anchor proteins are often involved in directing cells. Can the cells move these?
Yes.
What process is being described here? Proteins are made fluoresce. A certain region has this removed via bleaching. Monitored how long it takes for the fluoresce to come back. Some will not.
FRAP.
Why will some of the fluorescence not return in FRAP?
As the proteins are fixed.
What did Atomic Force Microscopy show in regards to the membrane surface?
Showed that the membrane was pretty flat with membrane proteins sticking up out of it.
What is an example of an organelle with lots of membrane proteins?
Mitochondria.
Why is it a disadvantage for an organelle to have lots of surface proteins?
Barrier to movement.
What is the issue with he fluid mosaic model?
It concentrates on proteins and not lipids.
What two other things can a phospholipid be called?
A phosphoglycerides or glycerolipids.
What type of double bonds are often found in phospholipids?
Cis.
What type of bonds are often found in sphingolipids?
Trans.
What is present instead of O in sphingolipids?
NH.
Name one example of a sterol.
Cholesterol.
Sterols can be described as what compared to other membrane proteins?
Shorter and fatter.
What type of double bonds do membranes prefer due to their lower energy?
Trans.
How high can the molar % of cholesterol be in some membranes?
Up to 50%.
What shape is cholesterol?
Fairly flat.
Is cholesterol polar?
Yes but it does not contain a polar head group, it only contains one polar OH.
Archae membranes are similar to those found in prokaryotes and eukaryotes?
False they are completely different.
What bonds are found in the fluid phase of the membrane?
Cis bonds as they are more flexible.
What bonds are found in the gel phase of the membrane?
Trans bonds.
What stage does the cell want the membrane to be?
Fluid.
How can you change from the gel phase to fluid phase?
Heat. The change is sudden.
Apart from regulating the fluidity what else does changing the phase of the membrane from gel to fluid do?
Makes the membrane thinner.
What does patch clamping measure?
Change of current in a membrane due to the flow of ions.
Describe the basic process of patch clamping.
The glass electrode is pulled to a very small point on the membrane and tiny currents are measured between the two electrodes. The pipette pushing on the cell is called the on cell positioning. A very tight seal is formed between the tip and the membrane so nothing leaks.
What does ‘Inside out’ geometry refer to?
What was inside the cell corresponds to whats outside the tip.
Why is it good that patch clamping involves a very small surface area of the membrane?
As only a very small number of channels will be present. Allows you to study the channels individually.
What does ‘outside out’ geometry used for?
To study external ligands linked with channels.
What do voltage gates across a membrane cause?
Channels to open.
How much current is present across the membrane when the channel is closed?
0.
What charge value corresponds to a channel being open?
5pa.
Do all channels have the same current when open?
Yes.
What two types of channels are present only axons of nerve cells?
Na+ specific and k+ specific.
What is the resting voltage of an axon membrane?
-70mv.
What happens when the axons membrane potential reaches 0?
Maximum ion flow.
When does the plug attach to the voltage gated Na+ channel ?
1ms. It will detach a few sections after the membrane potential has returned to normal.
What does ‘whole cell geometry’ in patch clamping allow?
Allow the cells total response to be monitored.
What is the voltage gated Na+ plug made from?
4 identical a helices with positive residues.
Why can the plus on the Na+ voltage pump close after depolarisation has occurred?
The plug is made of positive helices. When the cell depolarises and becomes more positive the plug will move to the positive side of the membrane.
What way does Na+ flow through the axons Na+ voltage gated channel?
Either way.
When is the axon said to be refracting?
When the plug is in the whole.
What is the main difference between the Na+ voltage gated channel and the K+ voltage gated channel in the axon?
The K+ channel opens and closes slower.
What is the K+ voltage channel of the axon also called?
The delayed K+ channel.
When the current is bigger why is it easier to driveK+ through the voltage gated axon channel?
The difference in charge is greater meaning it is easier to drive the positive ion through. THE CHANNEL DOES NOT OPEN MORE.
What two pumps are present in the membrane of all cells?
- Na+/K+ pump. 2. Resting K+ channel (K+ lack channel.)
What does the Na+/ K+ pump require?
ATP.
What way do the Na+ and K+ ions flow in the Na+/K+ channel?
Na+ out K+ in.
What is the concentration of Na+ on either side of the Na+/K+ pump?
150mM exterior and 12mM cytosol.
What is the concentration of K+ on either side of the Na+/K+ pump?
4mM exterior, 140mM cytosol.
When is the resting K+ channel open?
All the time.
What channel is responsible for the cells negative potential?
Resting K channel.
The resting K+ channel does not transport much K+ so it is not energetically costly. True or false?
False.
The K+ leak is the only channel in the axon open at rest. Why?
It tries to neutralise the K+ concentration, causing a negative charge inside the axon. (The cell does not have enough energy to completely neutralise this charge.)
What is the difference between the motor neurone and the sensory neurone?
The motor neurone brings nerve signals from the brain to the muscle and the sensory neurone does the reverse.
Nerve impulses make an all or nothing response. How is a stronger signal obtained?
More frequent action potentials.
Is there attenuation of the nerve signal as you travel down the axon?
No.
How frequent are nerve impulses at their maximum?
4 m/s.
Are resting K+ channels voltage gated?
No.
How much ATP does the Na+/K+ pump use?
25%.
The resting K+ channel causes the membrane to slowly depolarise. At what voltage does the Na+ gated channel open?
-40mv.
The Na+ voltage gated channel opens at -40mv. What way do the Na+ ions move?
In due to the concentration gradient. This causes the axon to depolarise more.
The opening of the Na+ channel causes the voltage of the membrane to go up to what?
+35mv.
What type of feedback is found in the axon?
Postive.
When does the Na+ voltage gate get plugged in the transmission of a nerve impulse?
When the membrane potential reaches +35mv.
When does the delayed K+ channel open in the transmission of an action potential?
Once the membrane potential has reached +35mv and the Na channel has closed. This brings the membrane potential back to normal.
Why does the membrane slightly hyperpolarise in neurotransmission?
As there is a delay in shutting the K+ voltage gated channel.
Why is there a refractory period in neurotransmission?
As the Na+ plug stops the Na+ channel opening immediately.
Why can the action potential only go one way along the axon?
Refractory period is present.
At neuronal junctions, such as in the brain a logic gate is present. What is a logic gate?
A signal can produce either a positive or negative response which can add up. An action potential in the scone neurone will only be started once the net voltage at the axon hillock reaches a certain level.
How much Na+ moves into the cell/ how much K+ moves out of the cell with each action potential per um2 of the membrane?
10^5.
What percentage of Na+ and K+ in the cell is involved in the Na+/K+ pump?
1%. Neurones can transmit multiple signals without effect on the Na+ and K+ concentrations of the cell.
How fast does an action potential travel without a myelin sheath?
1m/s.
How may times faster does the myelin sheath make action potentials?
1000X.
How large are the gaps on the nodes and how regular are they?
1um and every 100um.
At the nodes what does the membrane contact?
Extracellular fluid.
What is caused by loss of myelin in some areas of the brain and spinal cord?
MS.
Can real and artificial membranes be altered by heat?
Yes.
What three things does lipid composition influence in a membrane?
- Fluidity. 2. Shape. 3. Thickness.
When cholesterol is at a high concentration in the membrane what does it do?
Make the membrane flatter.
What bond causes disorder resulting in shorter membranes?
Cis.
What does cholesterol do to SM trans bonds?
Makes them longer and with it thicker. .
Do membranes have different types of local thickness?
Yes.
What does the head shape of PC cause?
The membrane to all line up as the cross sectional area of the tail and the head group are the same.
Why does PE have a much smaller head?
It only has a H in place of a methyl group.
What does PE’s head shape result in?
The membrane folds up so the heads are closer together. This prevents holes.
What is special about the head group of PI?
It is much bigger.
What does the large head group of the PI cause?
The membrane to curve.
What 5 things can the membrane control to alter the curvature?
- Location of lipids. 2. Helix insertion into one side. 3. Membrane proteins. 4. Cytoskeleton. 5. Scaffolding of proteins.
Where are direct proteins scaffolds found?
Inside and out.
Where are indirect protein scaffolds found?
Inside.
Where is positive direct scaffolding found?
Outside.
Where is negative direct scaffolding found?
Inside.
In what membrane is there more SM and cholesterol?
Outer membrane.
All GPI anchored proteins are found on the outside. True or false?
True.
All lipid anchored proteins are found on the outside. True or false?
False.
Why can lipids not flip themselves in a membrane?
It requires too much energy.
What ATP dependant enzyme is needed to flip a membrane lipid?
Flipase.
Describe phase separation.
Different lipids components separate within a membrane due to their different compositions of lipids and because of the fact that each component likes to associate with itself. The membrane will want to do this spontaneously but can also encourage this.
Why does the fluid mosaic model not describe the membrane properly?
Phase separation
A lipid raft is a thicker region of the membrane. What is it made of?
Cholesterol and sphingolipids.
Where do proteins with longer transmembrane helices go?
Lipid rafts.
Where do proteins with GPI anchors go?
Lipid rafts.
Where do proteins with palmitoyl anchors go?
Lipid rafts.
What sort of proteins prefer not to be in lipid rafts?
Proteins with prenyl anchors.
Does phase separation happen to lipid rafts?
Yes.
Is the formation of lipid rafts spontaneous?
No.
The formation of lipid rafts is controlled by the cell. Why is this beneficial for the altering the location of membrane proteins (3 reasons.)
- Bringing signal systems together. 2. Organising the start of endocytosis. 3. T cell activation.
What did AFM show in regards to lipid rafts?
They were much thicker than other parts of the membrane.
What can sometimes form when a cell is stimulated?
A large, stable lipid raft.
Why can a lipid anchor be moved?
It is just an ester bond that can be moved with covalent modification.
Proteins are tagged allowing localisation in a cell. Is the membrane vesicles also tagged?
Yes.
Can lipid components of the membrane regulate targeting?
Yes.
What brings about ligand-mediated endocytosis?
Lipid rafts.
What protein binds to the lipid raft formed for ligand-mediated endocytosis allowing it to bend?
Caveloin.
What two proteins can caveloin recruit?
Cavin and clathrin.
When will a lipid raft be small and short lived?
When it is unstimulated.
What two functions do lipid rafts have?
- Signalling. 2. Endocytic pits.
What are the three main functions of ion pumps and channels?
- Biological energy conversions. 2. Signal transmission in the NS. 3. Muscle activity.
Membranes can be c_____, c______ and m______.
Carriers, converters, markers.
What are the three types of membrane proteins?
- Integral. 2. Peripheral. 3. Lipid anchored.
How many anchors can a lipid anchor protein have?
Multiple.
How many leaflets of the membrane are lipid anchor proteins attached to?
One.
How do peripheral anchor proteins attach to the membrane?
Non covalently. This can either be through VDV/ hydrophobic interactions or through non covent interactions with other membrane proteins.
What do membrane spanning regions of membrane proteins require?
All main chain hydrogen bonds to be made as the inside of the membrane is hydrophobic.
Even when all the H bonds have formed the transmembrane helix can still be described as polar. True or false?
False.
What is hydropathy?
Free energy for the transfer of an amino acid residue in an alpha helix from the membrane interior to the water.
What does a higher hydropathy value mean?
A more hydrophobic residue.
What amino acid is at -4.5 on the Kyte and Doolittle scale?
Arg.
What amino acid is at 4.5 on the Kyte and Doolittle scale?
Ile (isoleucine.)
The membrane is 3.5nm. How many amino acids need to be in the transmembrane helix to span the whole membrane vertically?
20.
A window of how many residues is used to calculate hydropathy?
19 residues.
What is sometimes added to the transmembrane helix?
Hydrophilic residues to give extra function to the helix.
If a helix is diagonal in the membrane how many residues could it require?
36.
Beta barrels can fit into the membrane but beta sheets can not. Why?
Because not all man chain hydrogen bonds are made.
What is an omp?
An outer membrane protein.
What is the sequence pattern in beta barrels?
Alternating hydrophobic and hydrophilic.
Why can you not use the Kyte and Doolittle scale with beta barrels?
As the sequence alternates between hydrophobic and hydrophilic residues.
What value does an amino acid need to have on the Kyte and Doolittle scale for it to be part of a transmembrane helix?
1.6.
What four linkages can be made by a lipid anchor protein?
- Acetlylaiton. 2. Prenylation. 3. Thioester. 4. GPI anchor.
What is myristoylation?
An amide bond made to the N terminal glycine.
Myristoylation is an example of acetylation. When does it occur?
Co transitionally after the N terminal met has been removed.
Is acetylation reversible?
No.
What is prenylation?
When a thioETHER linkage is formed with a C terminal cysteine.
What is the difference between a thioether and a thioester linkage?
A thioether linkage contains a S instead of an O.
Does prenylation involve a terminal cysteine?
No it doesn’t have to. Cys-a-a-x can be bound too. The a-a-x is cleaved (a represents a aliphatic amino acid.)
Where do thioester bonds form in regards to lipid anchors?
With any cysteine in the chain.
There are no nonsense sequences involved with thirster sequences in lipid anchors, however they have a higher tendency to be found where?
Close to prenylation and acylation sites.
What enzyme makes thioester bond formation reversible?
Thioesterases.
What 5 things is signalling a response for?
- Hormones. 2. Growth Factors. 3. Infections. 4. Neural synapses. 5. Bacterial response to the environment.
What are two examples of short term signalling?
Vison and pain.
What are two examples of long term signalling?
Cell differentiation/ sex hormones.
What two things do external signals normal effect inside the cell?
- Enzymes. 2. Gene transcription.
What four ways can an external signal alter inside the cell?
- Hydrophobic molecule to an intracellular receptor e.g. NO.
- Ion through an ion channel e.g. post synaptic receptors.
- Ligand to an enzyme (normally a kinase.)
- Ligand to a G protein coupled receptor.
Signalling can involve a kinase enzyme. These can only ever be on or off. True or false?
True.
When is a G protein on?
When it is bound to GTP.
When is a G protein off?
When it is bound to GDP.
What is RAS an example of?
A G protein.
What residue is found in switch one of Ras?
Thr35.
What residue is found in switch two of Ras?
Gly60.
How much more GTP is in a cell compared to GDP?
10 times.
What does Guanine exchange factor do?
Allow GDP out of RAS and GTP in.
What protein helps RAS hydrolyse GTP?
GTPase activating protein.
When is a kinase activated?
When it is phosphorylated.
What mechanism gets a signal across a membrane with receptor- linked kinases?
Dimerisation.
What does dimerisation of a receptor linked kinase cause?
2 domains next to each other in the cell to autophosphorylate.
What does a phosphorylated receptor act as?
A binding site for modular adaptor proteins including Grb2 and Sos.
One kinase domain will be phopsohorylated by the other. What does this do?
Fixes the position of the activation loop allowing the substrate to bind correctly.
What are two examples of modular adaptor proteins?
Grb2 and SOS.
What two regions is the Grb2 modular adaptor protein made of and how many of each region does it contain?
1 SH2 region and 2 SH3 regions.
What do the SH3 domains of Grb2 recognise?
Polyproline helices.
What does the SH2 domain of Grb2 recognise?
Phosphotyrosines.
Is there a specific SH2 or SH3 for each receptor?
SH3.
What part of the SOS modular adaptor protein binds to SH3?
Proline rich arm.
What two things come together to activate G protein RAS?
SOS and Grb modular adaptor proteins.
What is the main function of the G protein Ras?
To activate Raf.
Was does Raf activate?
A chain of kinases.
What is an example of a Ras activated signal pathway?
Rad-Raf-Mek-Erk.
What is Erk and what does it do?
It is activated by a cascade activated by Ras which phosphorylates several transcription factors. This moves into the nucleus and alters the serum response element which is responsible in controlling early response genes.
Using the G protein Ras pathway is a complicated way to get a signal to the nucleus. What are its benefits?
It allows the signal to be amplified at each step and for modification of the signal.
What do GPCRs do?
Link the signal more directly with the G protein.
What percentage of drugs in clinical use are targeted to GPCRs?
50%.
What are GPCRs made from?
7 transmembrane helices.
GPCRs are made of 7 transmembrane helices. Between which of these helices is the intracellular loop found?
Between 5 and 6.
What does the intercellular loop of GPCRs bind to?
A hetereotomic G protein.
What three units are found in a heterotromic G protein?
Alpha Gbeta Ggamma.
What two units of the heterotromic G protein are always bound?
Beta and gamma.
Where does the ligand bind to in GPCRs?
Well within the membrane.
What is an example of a GPCR?
Rhodopsin.
What does rhodopsin respond to?
Light.
What amino acid is the toggle switch in rhodopsin made from?
Tryptophan.
Describe the process used in rhodopsin.
Binding of a ligand moves the tryptophan causing the bent helix in the rhodopsin to rotate. This changes the conformation of loop 3 which opens up a groove allows Ga to bind. A helix from Ga fits into the groove, opening up the binding site in Ga allow GDP to leave.
What part of the SOS modular protein activates RAS?
The GEF domain.
What can a receptor bound GPCR act as?
A GEF turning on the signal.
What is a common target for Ga-GTP?
Adenyl cyclase.
What does Adenyl cyclase do?
Coverts ATP to cAMP when it is bound to Ga-GTP.
What is Ga its own?
GAP meaning that it can hydrolyse the GTP to turn off the signal.
What are ion channels really responsive to?
Ligand concentration.
Ion channels are made of multiple subunits. How many are there normally?
5
What do the subunits of the ion channel do?
Simultaneously rotate, work like an ‘iris of a camera.’
One type of lipid anchor is the GPI anchor. What does GPI mean?
Glycosyl phosphatdylinositol anchor.
What does the GPI linkage contain?
Modify C-terminus with ethanolamine linked to oligosaccharide linked to inositol of phosphaldyl inosito.l
What two linkages are found in bacterial lipid anchors?
Thioether and amide linkages.
In what lipid anchor is the lipobox sequence found?
Bacterial.
What is the lipobox sequence?
[Lvi]-[AStvi]-[Gas]-C.
The lipoprotein signal sequence in bacteria is made up of a general region an a tripartite region. What is the tripartite region made off?
Postive N region, Hydrophobic region, Lipobox with invariant cysteine.
How many amino acids is the n region of the tripartite region made of and how many of these are positive?
5-7 residues. 2 positive (R or K).
How long is the hydrophobic region of the tripartite sequence?
7-22 residues.
What amino acid is found at the N terminal of bacterial lipid anchors?
Met.
