Lectures 1-6 (membrane structure & function) Flashcards
What are the equations for Ohm’s law?
Current (I) = Volts (V) / Resistance (R)
Current (I) = Volts (V) x Conductance (G)
What is Conductance?
G
1/Resistance
What is the Nernst equation?
cant write here
What do each part of the Nernst equation stand for?
R F T z
R - Gas constant 8.314 V C K-1 mol-1
F - Faraday’s constant 9.648x104 C mol-1
T - Absolute Temperature (°K) 273.0°K at 0°C
z - Ion valency (charge) ±1,2,3…
what happens when [Ion]o = [Ion]i
Eion = o
because x ln1 = o
what happens when [Ion]o > [Ion]i
= > 0 if z is +ve
=
what happens when [Ion]o
= if z is -ve
what do all cells do/have?
have a membrane potential and express ion channels
what are integral and peripheral proteins?
integral embedded in the membrane.
peripheral one side or the other.
Describe the Fluid Mosiac Model of the lipid bilayer.
Singer and Nicholson 1972.
Emphasizes fluidity, simplicity.
lipid bilayer as a consequence of a series of random electrostatic forces, causes them to line up.
There are some proteins floating around, don’t touch anything.
What replaced the Fluid Mosaic Model?
the evolved Fluid mosaic model emphasises order.
“the picture of a membrane as a lipid sea in which proteins float freely is greatly oversimplified”…
what are saturated/unsaturated lipids?
saturated - no double bonds
unsaturated - 1-6 double bonds
Long chains = less fluid
Cis/trans unsaturated bonds?
Cis kinked.
Double cis bonds make membranes fluid (kinks stop lipids packing closely together)
Trans found in processed foods, increased cholesterol.
describe the types of membrane lipids.
PHOSPHOLIPIDS, all have a glycerol backbone.
Phosphatidylethanolamine, Phosphatidylinositol, Phosphatidylserine (if on outside signals phagocytosis) and Phosphatidylcholine.
GLYCOLIPIDS
Galactocerebrosides and Gangliosides
CHOLESTEROL
how are lipids distributed?
asymmetrically, generated by lipid flippase ATPase pumps.
Move lipids from one leaflet to the other leaflet?
What happens if there is a loss of symmetry?
inhibition of flippases and activation of scramblases.
PS presented on the outside of the cell, signalls macrophages to start phagocytosis/apoptosis.
what is the role of phospholipids?
structure signalling (PI)
what is the role of cholestrol?
maintaining stability.
decreasing permeability.
what is the role of glycolipids?
cell recognition.
protection, additional layer on outside of cell.
can influence electrical properties of the cell.
describe the ratio/contribution of lipids and proteins.
50 lipids : 1 protein
but proteins contribute 30-45% mass since much larger.
what is a GPI anchor?
anchors protein in the outer leaflet of PM.
Specific phospholipase may release this protein from the cell as a signal pathway.
Describe the structure of transmembrane protein structures
Polar protein regions:
- Prefer polar environment
(H2O + lipid headgroups)
- Loathe lipid interior
Non-polar regions:
- Prefer non-polar (bilayer)
- Loathe H2O + lipid heads
describe membrane synthesis.
Membrane lipids synthesised in ER, then “randomly” incorporated into ER membrane.
Lipids trafficked in membrane vesicles which fuse in to plasma membrane.
Lipid asymmetry established by flippases.
Membrane proteins are synthesised at RER and trafficked to the plasma membrane.
what is SRP?
signal recognition particle..
translocator allows the protein to work through the membrane and come through the other side.
For secreted protein, once protein has gone through translocator,
SRP can be cleaved and you get a free floating soluble protein.
Describe how SRPs work for transmembrane proteins
Single span/pass protein:
need a start and stop transfer sequence.
stop sequence the a helix, causes protein to stay in the membrane.
what can proteins do in a membrane?
what can’t they do?
can spin about z axis,
can change shape,
can move laterally.
can’t translocate vertically,
can’t rotate.
how can cells control the diffusion in membranes?
aggregation of cells/proteins, less space to move/interact.
Tethering to macromolecules outside the cell (e.g. extracellular matrix).
Tethering to macromolecules inside the cell (e.g. cytoskeleton).
Interaction with molecules on adjacent cells.
Barriers e.g. Tight junctions - epithelia.
Axon /soma junction (initial segment) – nerves.
describe lipid rafts.
aggregations of proteins and lipids from the membrane.
segregation and stabilization of them.
high conc of cholesterol and sat fatty acids (allows tight packing).
GPI and FA anchors.
membrane is slightly thicker, longer chains.
why are lipid rafts important?
Protein trafficking to membrane.
Signaling complexes : integrins, GPCRs, channels, etc
Disease - target for viruses, bacteria, prions, parasites
what is overton’s rule?
solubility of a substance in oil is proportional to its bilayer permeability (Pbilayer).
not completely true, membranes have a range of permeabilities.
how does the rate of transport for facilitated diffusion and simple differ?
facilitated has a saturation point (graph of rootx), simple has a graph of y=x.
describe ion channels.
facilitated diffusion for ions.
high rates of transport.
very diverse.
what are carrier mediated transport proteins?
Transport of more than one substrate (at least one down gradient and one may be against gradient).
Extremely diverse range of substrates.
can exist as cotransporters/symports, exchangers/antiports
name 2 cotransporters.
Na+,2Cl-,K+ cotransport (NKCC1)
Na+-glucose cotransport (SGLT1) intestine/kidney.
what is a cotransporter/symport?
two or more substrates (ions, organic solute) transported in the same direction.
what is an exchanger/antiport?
two or more substrates (usually ions) transported in opposite directions
give 2 examples of exchangers.
Na+ H+ exchange (NHE)
Cl–HCO3- exchange (AE)
describe ion pumps.
Transport of one or more substrate (at least one against gradient).
Pumps have enzymatic activity (ATPase) to hydrolyze ATP to release energy for ion transport (PRIMARY active transport).
what influences the transport of ions across a membrane?
chemical gradient/membrane potential.
describe the electrochemical gradient.
electrical (mV) and chemical (mM) influences.
EC gradient = Vm - Ex (eq potential/nernst).
what is the function of the nernst equation?
The Nernst equation “converts” a chemical gradient in mM to an electrical gradient in mV
what substances don’t have the membrane permeability we would expect?
water and urea
how can the permeability of the lipid bilayer be changed?
cholesterol.
increased cholesterol = decrease permeability to water and gases.
membrane permeability to H2O/maybe gases is mediated by aquaporins.
what are aquaporins?
Membrane permeability to H2O (and possibly gasses) is mediated via water channels called Aquaporins.
e.g. kidney collecting duct normally impermeable to H2O – BUT insertion of AQP2 PH2O
what is the main aquaporin?
AQP4 - main CNS channel.
knock out in mice, increases survival frmo middle cerebral artery occlusion.
decreases brain inflammation.
AQP4 blockers sought to treat inflammation.
how is electroneutrality maintained in a cell?
there is a difference between the totals for [C+]i (~136mM) and [A-]i (~40mM), but electroneutrality is maintained by “fixed”-ve charge on intracellular proteins.
what is Vm?
membrane potential
how is cell volume maintained?
through gradients.
cell membrane permeable to water, osmosis can threaten cell volume. (osmoregulation).
how do extracellular osmolarity and intracellular osmolarity relate?
usually the same.
however a change in extra or intracellular osmolarity causes the movement of H2O by osmosis → change in cell volume
What is the pump leak hypothesis?
wrong.
K/Na pumps.
resting K permeability.
pump K in as it leaks out.
Cl has a key role. Can go either way across the membrane.
??
what are accumulators and extruders?
accumulators send ions into the cell, extruders out.
what happens in a hypertonic solution to a cell?
increased extracellular osmolarity. (towards salt)
H2O leaves the cell, the cell volume decreases.
what happens in a hypotonic solution in a cell?
Decrease in extracellular osmolarity.
H2O enters the cell, volume increases.
why do volume changes in a cell not reach the theoretical values?
Osmotically inactive volume = 15-30% does not respond to osmotic challenge.
Cell volume regulatory mechanisms are activated in most cells before full volume change is complete!
Describe RVI
regulatory volume increase.
Net Gain of K+ and Cl- →
H2O follows by osmosis.
NKCC1 cotransporter (Na, 2Cl, K in) Na moving down gradient, Cl and K against their electrochem gradient.
NHE1 and AE2 antiports:
NHE1 - Na in H out
AE2 - Cl in HCO3 out.
functionally coupled, when NHE1 pumps it activates AE2.
in both cases driven by Na gradient, BUT Na+ which enters is removed by Na+, K+ ATPase (maintaining Na+ gradient) net gain of K+ (pump-leak).
Both mechanisms generally present, and thus Cl- “accumulated” in most cells.
describe RVD
regulatory volume decrease.
Loss of K+ and Cl- →
H2O follows by osmosis
K/Cl cotransporter.
K+ leaves, drives Cl out with it.
most cells rely on ion channels - faster.
K functionally coupled to Cl channels.
Transport driven by K+ and/or Cl- gradient.
Generally only one of two mechanisms for RVD.
Most cells use ion channels.
Select group of “atypical cells” use the cotransporter.
what “atypical” cells use cotransporters for RVD?
mature neurons, pancreatic a-cells
low intracellular Cl conc, opening of Cl channels causes Cl to flood in, not helping high cell vol.
In these cells Cl- is not accumulated so no gradient for Cl- exit (KCC responsible for RVD using K+ gradient). In neurons (& α-cells) Cl- is actively extruded by “constitutively” (happening all the time) active K+-Cl- cotransporters.
Opening of Cl- channels causes Cl- influx and results in the hyperpolarisation of Vm.
(This is the basis of GABAA and glycine receptor synaptic inhibition (both are Cl- channels))
describe RVD
regulatory volume decrease.
Loss of K+ and Cl- →
H2O follows by osmosis
what “atypical” cells use cotransporters for RVD?
mature neurons, pancreatic a-cells
low intracellular Cl conc, opening of Cl channels causes Cl to flood in, not helping high cell vol.
why do most cells have the ability to volume regulate?
developmental role, maintaining cell volume following cell division and/or during cell migration.
describe how cell volume influences the brain tumour metastasis.
tumours ↓ cell volume (loss of K+ and Cl-) allows glioma cells to squeeze between other cells in the CNS during metastasis, then regain once through.
Chlorotoxin (a Cl- channel blocker isolated from scorpion toxin) inhibits glioma cell migration in vitro.
In vivo fluorescently labelled chlorotoxin now being used to identify tumour cells during surgery.
how do we change cell volume?
cant change extracellular osmolarity unless kidneys r fucked.
change intracellular osmolarity.
why do most cells have the ability to volume regulate?
developmental role, maintaining cell volume following cell division and/or during cell migration
describe how cell volume influences the brain tumour metastasis.
↓ cell volume (loss of K+ and Cl-) allows glioma cells to squeeze between other cells in the CNS during metastasis.
Chlorotoxin (a Cl- channel blocker isolated from scorpion toxin) inhibits glioma cell migration in vitro.
In vivo fluorescently labelled chlorotoxin now being used to identify tumour cells during surgery.
what does pH = ?
-log10 [H+]
why is pH not useful in physiological studies?
what do they do instead?
narrow range in living cells.
stats are more complex on logs
often quote [H+] in nM
how is pH measured?
pH sensitive fluorescent indicators.
Cells incubated with lipid permeable ester of the indicator.
Indicator ester diffuses into cell, is hydrolysed to ionic form and is trapped in cytoplasm.
Indicator is excited
(dual excitation = controls for dye loss via leak or photo-bleaching).
Fluorescence is emitted.
Calibrate → pHi
when is best to measure the pH?
when equilibrium is disturbed.
what is an acid extruder?
ie Na+ -HCO3-
drives H+ out by taking Na in.
???
what are acid loaders?
take OH- out of the cell.
why must Ca i be so low?
due to phosphate ions, they form insolute precipitates with Ca (ie bones/teeth).
how is ca i kept so low?
extrusion across cell membrane: NCX - Ca out 3Na in PMCA Ca out H in, uses ATP. SERCA 2H out 2Ca in, ATP. Ca accumulates in SR/ER
sequestration into organelles:
Ca entering slows release from Ca storage???
what is electroneutral?
no net movement of charge
what is electrogenic?
net movement of charge generating a small electrical current
how can the same Ca signal have different responses?
temporal differences/speed
spatial differences
what barriers does our body have?
Skin
Intestines, airways, reproductive tract
exocrine glands, kidneys, liver
“meninges” (Greek for membrane) around the brain
Blood-brain, Blood-testes, blood-retina, placenta
most are involved in transport, not just barriers.
how are epithelial cells linked together?
junctional complexes made up of claudins and occludins.
23 different claudins allow paracellular transport, determines tightness/selectivity of junctions.
what is paracellular transport?
between cells
what is transcellular transport?
across the cell, must cross multiple membranes. needs to be polar?
describe the stages of salivary secretion.
primary secretion: ions from blood to lumen, h2o follows by osmosis through aquaporins.
secondary modification: reabsorb Na and Cl but not H2o, there’s no aquaporins.
final saliva is hypotonic compared to plasma (decreased NaCl)
describe acinar secretion
basolateral side:
Na pump puts 3Na out and 2K in. generates a Na gradient. primary active transport ATP.
K leaks out
K2ClK cotransporter brings in Cl. Secondary active transport.
Cl K move against gradient due to Na gradient
net accumulation of Cl inside the cell.
- membrane potential inside and Cl driven in from basolateral side drives it out through the apical side.
claudin 2 paracellular movement of Na from basolateral side through to apical side.
what other phenomena affect acinar cells?
split up into single cards
basolateral side has ACh receptors which increase IP3 which leads to an increase in [Ca]i.
Ca activated channels (Cl on apical and K on basolateral side).
loss of K/Cl leads to decrease in volume since water is drawn out via osmosis.
HCO3- efflux leads to increase in [H+]
co-transmission: ACh
main parasympathetic NT?
ACh
main sympathetic NT?
Noradrenaline
what is VIP?
released by parasymp
??
describe the barriers of the brain.
2 layers of dura mater
superior saggital sinus allows Cerebrial spinal fluid to drain out of CNS into normal vascular system.
arachnoid membrane
subarachnoid space filled with CSF
blood brain barrier made up of brain capillary endothelium, held in place by astrocyte cells
where is CSF made?
choroid plexus’s
describe the BBB
2 parts
protect the brain.
made of endothelial cells of brain capillaries.
second interface: in choroid plexus’s make up blood-CSF barrier.
engage in transport to provide nutrients and remove waste.
describe the choroid plexus barrier.
epithelial cells linked by junction complexes.
describe the brain endothelial cell barrier
capillaries lined by endothelial cells, have adopted an epithelial morphology (polarised) and linked by junctional complexes, very tight.
contain many transporters.
what transporters are present in the BBB?
A variety of organic cation and anion transporters (OATPs and OCTs)
A number of specialised pumps (ABC proteins, e.g. p-glycoprotein)
Transporters expressed in liver, kidney and BBB
Role in transporting endogenous substances, e.g. OATPs = bile acids, steroid metabolites.
BUT also transport xenobiotics (drugs & toxins) e.g. p-glycoprotein = ABC protein “pump” for lipid soluble compunds (associated with multi-drug resistance in tumours)
what is p-glycoprotein?
ABC protein “pump” for lipid soluble compunds (associated with multi-drug resistance in tumours).
describe anti-histamines.
thought decreased lipid permeability = less CNS action = not crossing BBB
WRONG
1st gen ie chlorphenamine - sedative effects since acts on CNS
2nd gen non sedative effects as less action on CNS.
more lipid soluble.
both cross BBB
2nd gen better substrates for p-glycoprotein so are removed from CNS better
describe ivermectin
drug used to treat parasitic worms.
lipid soluble so crosses BBB but is then removed by p-glyocprotein.
mutation in collies reduces expression of p-glyco so can’t give to them. won’t be removed and can be neurotoxic.
