Williamson (Biological functions of membranes) Flashcards
What are the functions of membranes from an evolutionary perspective?
- arose as barrier between controlled env of inside and outside (stops content leaking out and random chemicals from coming in)
- permit and reg transport of nutrients (and waste) = CHANNELS
- dev ability to do against conc grad = PUMPS
- all cells do this, “tacked on” to original role of barrier
What are the later developments in the functions of membranes?
- conversion of membrane pot to energy (most cells)
- cellular recognition (euks and proks, but differently)
- signalling from outside to inside (all cells but no universal system)
- movement of molecules w/in euk cell in vesicles
- compartmentalisation (only euks)
How do cell sizes vary?
- E. Coli ≈ 2μm x 1μm
- epithelial cell ≈ 10x bigger each way
- fibroblast ≈ 4x larger width and breadth
- nerve axon = up to 500,000x longer
- vol of euk cell ≈1000x greater than prok, so vital need to compartmentalise and direct molecules appropriately
Where are almost all important functions w/in euk cell contained?
- membrane bound vesicles
How does internal membranes SA compare to external?
- internal SA 10x longer than external
What are membranes made up of?
- lipids
- hydrophobic proteins (prod fluid mosaic structure
- integral membrane protein
- peripheral membrane proteins
- lipid anchored proteins
How do lipids aggregate?
- spontaneously in water
- in lab can aggregate into many diff structures (bilayer, liposome, vesicle) but only into bilayers in cells
How can lateral mobility of proteins be detected?
- FRAP (fluorescence recovery after photobleaching)
- membrane proteins labelled w/ fluorescent reagent
- bleach w/ laser, resulted in bleached area
- membrane proteins diffuse, resulting in fluorescence recovery
- doesn’t fully recover, ∴ not totally random lipid distribution
What does AFM (atomic force microscopy) show?
- shows height of diff components and proteins embedded in membrane sticking up above lipid bilayer
What is the big problem w/ the fluid mosaic model?
- concs on protein and assumes lipids more or less same
- they aren’t (don’t all completely diffuse freely in membrane)
What are the diff types of membrane lipids?
- main are phospholipids
- sphingolipids = contain NH instead of O and often trans double bonds instead of cis bonds found in phospholipids
- sterols (eg. cholesterol)
- sphingomyelins = mainly sat
- phosphatidylcholine (PC) = mainly unsat, so lipids in PC layers less linear and more disordered
How does lipid composition affect membrane fluidity?
- cis double bond forces chain to go off at angle
- trans can fit w/in linear extended chain
- ∴ bilayers containing cis bonds fairly disordered (fluid phase)
- bilayers w/ trans bonds more ordered (gel phase)
- cells normally want membranes to be fluid to allow movement w/in bilayer
- ∴ PC layers tend to be thinner as less ordered
Can bilayers change between phases?
- any real or artificial bilayer can be induced to change between phases by heating (gel –> fluid)
How does cholesterol affect membrane fluidity?
- at v high conc in some membranes (euks, esp mammals)
- flat so packs against other flat (trans) lipids and makes them even flatter and ∴ longer
- implying real biological membranes have idiff thicknesses depending on composition
Why are diff lipids diff shapes?
- so cells can control shape
- eg. PE headgroup smaller than lipid tail so makes bilayer curved
- PC basically cylindrical so packs well into flat bilayers
How can cells vary the curvature of membrane?
- lipid composition
- membrane protein oligomerisation
- cytoskeleton (cytoskeletal proteins push/pull membrane about)
- scaffolding = indirect (not directly attached, direct -ve (inside membrane) or direct +ve (outside membrane)
- amphipathic helix insertion
How do lipid concs vary between diff membranes?
- vary a lot
- ER, golgi and plasma membrane diff (pm has more cholesterol)
- carefully controlled by cell to give them diff properties
How does lipid distribution vary between 2 leaflets of membrane?
- sphingomyelin and PC mainly in outside (≈3/4)
- phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol (95%) mainly in inside
Are proteins in the membrane in 1 orientation?
- 100% in 1 orientation
- GPI anchored all outside
- lipid anchored all inside
What is the role of anchors in membranes?
- can be added or removed (and changed)
- anchors direct to diff membranes and diff parts of cells
- can direct proteins reversibly
What is flippase?
- ATP-dep enzyme
- flips lipids between bilayer leaflets (not spontaneous)
Why does phase separation of diff lipid components occur?
- to vary lipid composition
- creates diff regions w/in membrane
- thicker and more rigid regions richer in cholesterol and sphingolipids called membrane rafts
What are membrane rafts?
- rigid blocks diff from rest of membrane
How do proteins segregate into diff regions?
- proteins w/ longer transmembrane helices go into membrane rafts
- proteins w/ GPI anchors go into membrane rafts
- proteins w/ palmitoyl anchors go into membrane rafts
- proteins w/ prenyl anchors prefer not to be in membrane rafts
How are membrane rafts formed?
- controlled by cell and important mechanism for alt location of membrane proteins
- eg.
- -> bringing signalling systems together
- -> organising start of endocytosis
- -> T cell activation
What does AFM imaging of GPI anchored proteins in rafts show?
- model membrane made of dioleoylphosphatidylcholine
- also contains sphingomyelin, which collects into patches (these are thicker layers)
- contain GPI anchored protein, almost entirely found in rafts
How are rafts a good way to bring proteins together or to keep them apart?
- small rafts become larger rafts w/ stimulation –> so some brought close together and some further apart
- attachment of GPI anchor, prenylation etc. is covalent mod and can be easily alt
- ∴ many proteins can be easily moved in and out of rafts
- opp true = if proteins don’t want to be in raft, add tags to separate them
Where are lipid rafts thought to have important functions?
- signalling
- membrane trafficking
How do lipid rafts move proteins around?
- use membranes to do it
- proteins tagged w/ signal seq to direct them to right place
- typically proteins tagged, but so are membrane ‘parcels’ that contain them
- lots of recycling to make sure proteins end up where meant to be
- all tightly reg
- most tagging done by proteins, but lipid composition also reg targeting
- almost all movement along MT tracks
What is the process of ligand-mediated endocytosis?
- brought about by membrane rafts
- start w/ flat membrane, ligand binds to receptor and activates it
- formation of membrane raft
- proteins (caveolin) bind to membrane raft, insert halfway into membrane and make it curved
- caveolin recruits more proteins (cavin, clathrin), makes coat around caveolae (invagination)
- caveolae pinched off at top and move into cell
What is patch clamping and what did it show?
- attach v sensitive electrode to patch of membrane and measure current across membrane
- pipette filled w/ buffer and either applying small amount of suction (to get whole cell to measure all receptors
- -> by pulling (inside out to measure channels that open w/ internal binding
- -> or by suction then pulling (to outside out = most useful)
- found 2 ‘excited’ levels –> 1st level = 1 channel open and 2nd level = 2 open
- excited levels at fixed positions, shows all channels have same current when open
- opening and closing essentially random (indiv channels open for random amount of time) –> av opening and closing rates specific to channel
What are the types of ion channels present in axons?
- VG Na+ channel
- VG K+ channel
- Na+/K+ pump
- ‘resting’ K+ channel
What is the role of VG Na+ channels?
- channel starts to open at -40mV
- max ion flow when pot is 0 or +ve
- has ‘plug’ which closes after channel open ≈1ms, plug detaches few ms after membrane pot returned to normal
1) closed Na+ channel - initial depolarisation, movement of voltage sensing α helices, opening of channel (<0.1ms)
2) open Na+ channel - return of voltage sensing α helices to resting position, inactivation of channel (0.5-1ms)
3) inactive Na+ channel (refractory period)
4) repolarisation of membrane, displacement of channel - inactivating segment and closure of gate (slow, several ms)
What is the role of VG K+ channels?
- similar to Na+ channels, closed w/ -ve pot and open as pot gets less -ve
- main diff is opening/closer slower
- ∴ sometimes called ‘delayed’ K+ channel
- also has plug
What is the role of Na+/K+ pump?
- ATP dep
- constantly pumps 3Na+ out and 2K+ in
- maintains Na+/K+ concs inside cell that are v diff from extracellular concs
What is the role of resting K+ channels, and why is this important?
- ‘resting’ as open even when cell at rest
- allow K+ to leak all the time and gives cell membrane its -ve pot
WHY? - if membrane w/ no channels open and physiological K+ grad across membrane, K+ will rush out to equalise concs when K+ channels open
- leaving -ve charge inside and create +ve charge outside
- w/in short distance either side of membrane, K+ concs equal, held by charge separation across membrane
- at this point have stable situation, w/ -ve membrane pot
How do nerve impulses (action pots) work?
- neurons form network
- motor neurons have axons pointing from spinal cord to muscle
- sensory neurons point from tissue to spinal cord
- nerve impulse is transient change in membrane pot, “all or nothing”
- stronger signal obtained by more action pots
- can’t get closer than 4ms (refractory periods)
What happens during transmission of an action potential?
- at synapses, signal transmitted from 1 cell to next by neurotransmitters
- neurotransmitters stored in vesicles at end of axon
- arrival of AP triggers exocytosis of vesicle
- diffuse across synapse and bind to receptors
- opening of channel in postsynaptic membrane which activates signal
How do neural junctions differ from NMJs?
- at NMJ 1 AP = 1 transmitted signal, all that needs to happen
- at neural more complicated ‘logic gate’
- signal can prod +ve or -ve response, which add up
- -> AP only started in 2nd neuron when net voltage at axon hillock reaches certain threshold (input from many neurons)
How does K+/Na+ ration determine resting pot in all cells?
- -60mV pot across resting state cell membrane (inside more -ve)
- true in almost all human cells
- comes mainly from K+ flow out through resting K+ channels
- continually need to pump K+ in and Na+ out (K+/Na+ pump)
- ≈25% total ATP consumption used to power this pump
What is useful consequence of refractory period?
- action pot can only go forwards
How is an AP transmitted?
- AP moving along cell depolarises membrane
- enough to raise pot above -40mV which opens Na+ channels
- Na+ influx (large conc grad)
- makes pot more +ve and leads to more channels opening, +ve feedback and v rapidly all Na+ channels open
- Na+ influx and pot up to ≈35mV
- after 1ms ish, plug in Na+ channels closes all channels
- now at peak of AP
- delayed K+ channels start to open due to +ve pot
- allows K+ efflux and makes pot -ve again
- K+ flow enough to hyperpolarise membrane briefly
- plug in Na+ channel stops it opening for several ms (refractory period)
What effect do APs have on membrane and overall Na+/K+ concs?
- large effect on membrane
- little effect on overall concs (≈1% moved per μm2 of membrane
Do APs change in size as they travel down axon?
- same size
How can toxins affect APs?
- many work by blocking diff aspects
- eg. Japanese puffer fish poisonous due to tetrodotoxin which blocks VG Na+ channels
What is the role of myelin sheaths?
- APs travel at ≈1m/s
- allow up to 100x
- has gaps approx 1μm long every 100μm
- membrane only contacts extracellular fluid at these nodes
- allows pot to jump from 1 node to next
What causes MS?
- loss of myelin in some areas of brain and spinal cord
- eventually nervous system shuts down
Why is signalling vital for all cells?
- response to hormones, GFs, infection, neural synapses, bacterial response to env
How does signalling vary?
- ranges from v short term (vision, pain) to v long term (cellular differentiation)
- may need turning off, others constitutively expressed (sex hormones)
What are the major pathways for signals to enter the cell?
- receptor linked kinases
- G protein coupled receptors (GPCRs)
- ion channels
Which pathways for signals entering the cell use G proteins and what role do they play?
- receptor kinases and GPCRs
- eg. Ras
- turned on by dissociation of GDP and binding GTP, using GEFs (guanine exchange factors)
- turned off by hydrolysing GTP to GDP (simpler process), using GAPs (GTPase activating proteins)
How do receptor linked kinases work?
- hormone binds and receptor dimerises
- autophosphorylation
- phosphorylation of 1 kinase domain by other fixes position of activation loop, allowing it to bind substrate correctly
- phosphorylated receptor now the intracellular on signal –> acts as binding site for modular adaptor proteins
- SH3 domain recognises polyproline helices
- SH2 domain recognises phospotyrosines (specific SH2 for each receptor)
- plug together to bring GEF to cell surface
How does Ras perform its function now that it’s an active G protein?
- main function to activate kinase called raf
- raf at top of kinase cascade (seq of kinase that phosphorylate each other and amplify signal at each step
- MAPKKK –> MAPKK –> MAPK
- MAPK moves into nucleus and phosphorylates several TFs
- complicated but allows mod of signal in diff ways
Are GPCRs common drug targets?
- largest group of proteins used as drug targets
- used by half of current drugs
How do GPCRs work?
- 7 TM helices
- intracellular loops bind to heterotrimeric G protein
- ligand binds well w/in membrane
- heterotrimeric (3 diff subunits) –> β and γ always paired
- receptor bound GPCR acts as GEF and turns on signal
- Gα-GTP now active and moves along membrane looking for something to activate
- adenylyl cyclase common target
- converts ATP to cAMP when bound to Gα-GTP
- cAMP acts as 2nd messenger elsewhere in cell
- bound GTP rapidly hydrolysed to GDP by Gα, turning off signal (Gα is its own GAP)
What are some other targets of activated Gα?
- some G proteins indirectly open or close ion channels
- smell and vision work in this way
- binding of odorants to specific receptors activates Gα, activates adenylyl cyclase
- cAMP opens Na+ channels, initiating neuron depolarisation
- light leads to alteration in conc of cGMP (works in similar way)
- important class of GPCRs work by Gα activating phospholipase C (PLC)
How do ion channels work? (pathway for signals to enter cell?
- ligand gated channels so need to be v responsive to ligand conc
- may have multiple subunits (often 5)
- ACh receptor works by simultaneous rotation of each subunit to open up channel
