Williamson - Membranes Flashcards
what is a membrane raft, which phospholipids
thicker more rigid patch of membrane, contains more sphingolipids and cholesterol
what proteins associate with membrane rafts
those with long TM helices
GPI anchors
palmitoyl anchors
NOT prenylated anchors
how can you find membrane rafts
AFM (atomic force microscopy)
what can a membrane raft do
organise proteins
signal
trafficking - endocytosis
how does endocytosis occur
protein dimerises and brings lipid rafts together
caveolin proteins bind to one side (inner) causing a curve
more proteins recruited into coat
pinched at the top
what does patch clamping measure
the current across a membrane
what does inside out patch clamping geometry measure
current across the patch of inner leaflet membrane
what does outside out measure
current across outer leaflet membranes - useful for ligand gated channels - can put ligands in the solution
what does whole cell geometry patch clamping measure
current across the whole cell
what is the concentration of sodium inside and outside the cell
inside: 12mM
outside: 150 mM
what is the concentration of potassium inside and outside the cell
inside: 140 mM
outside 4 mM
when is the peak of action potential
when sodium channels close and potassium channels open
how does the Na+/K+ pump maintain the resting potential
it pumps 3 sodium out for every 2 potassium in maintaining ratio of sodium to potassium which is much lower that outside the the cell (against their concentration gradients)
inside: 150mM:4mM
outside: 12mM:140mM
what causes a cell to be hyperpolarised
a rush of K+ out of the cell
how would you characterise a voltage-gated channel
using whole cell patch clamping
vary voltage applied
and measure the currents
how would you characterise a ligand gated channel
using outside-out patch clamping
different ligands (and different concs) in the buffer solution
measure current
What K+ and Na+ channels/pumps are present in nerve cells
Na/K pump 3 Na+ for 2 K+
voltage gated Na+ channel –> action potential
voltage gated K+ channel –> repolarisation
K+ resting channel
what is the refractory period
a temporary period after an action potential when the membrane is unresponsive and unable to generate another action potential. This allows the cell to recover and ensures action potentials propagate in one direction.
Due to plug in sodium channel
4ms
what is hyperpolarisation
membrane potential is very negative (-70mV) making the cell less likely to generate an action potential
caused by resting K+ channel
what is repolarisation
when sodium channels are closed but potassium channels are open
membrane potential decreases from the peak (action potential)
what is depolarisation
change in cell membrane potential, making it more likely to generate an action potential
when the threshold (-55mV) is met and Na+ channels rapidly open - action potential (+30mV)
where does the energy come from to maintain the action potential along an axon
ATP powering the Na+/K+ pump
what are the structure features behind a voltage-gated channel
voltage sensing domains: eg. charged AAs, undergo structural changes to open or close the channel pore
selectivity filter for their ion
why is the potassium leak channel important
to maintain the resting potential
negative inside the cell
K+ can leave, leaving behind a negative charge
what is the equilibrium potential
the potential at which there is no net movement of an ion across a membrane
it is balanced by the electrical force pushing an ion one way and the concentration gradient pushing it the other
what does a myelin sheath do
insulate the axon
only allows action potentials to be transmitted at nodes = gaps in the sheath where Na+ channels are
travels 100x faster
saltatory conduction
what is a single pass G protein
Ras
what is a heterotrimeric G protein
the Gαβ𝛾 complex
Outline the mechanism of activation of the acetylcholine receptor
2 Ach molecules bind to the nAchR causing a conformational change in which the 5 subunits twist, opening the channel for ions
what is a second messenger
molecules which transmit signals from cell surface receptors to intracellular targets
intermediates between receptor and downstream effects and generated upon receptor activation
eg. cAMP, IP3, DAG
Gαs
stimulatory, ATP → cAMP → kinase cascade
Gαi
inhibits ATP → cAMP → kinase cascade pathway
Gαq
stimulatory, phospholipase C releases inositol headgroup from phosphatidylinositol, acts as a signalling molecule
What is a kinase cascade?
A sequence of kinases which phosphorylate each other leading to phosphorylation of a transcription factor, amplifies the signal
how can signals cross the cell membrane
ion channels, ligand or voltage gated
GPCRs
receptor linked kinases
hydrophobic ligands on cytoplasmic receptors
what does grb2 contain and what is it analogous to
an adapter
SH2 which binds a phosphate on a receptor linked kinase - specific end of adapter
2 SH3 domains which bind to proline-rich arm of Sos - general end of adapter (binds to general pathway)
what does Sos contain
a proline arm which binds the SH3 domains of grb2 and a GEF domain which promotes Ras GDP –> GTP –> signal
How is a GPCR turned off
receptor is phosphorylated by GRK
arrestin is recruited - turns off
receptor endocytosed, ligand removed, phosphate removed
cholera
ADP-ribose binds to Gαs
cAMP continuously made
Cl- out of CFTR –> diarrhoea
whooping cough
ADP-ribose binds to Gαi
inhibitory response turned off
too much cAMP made
high pKa
hydrophobic = protonated
