deck 1 Flashcards
patch clamp methods
whole cell
inside out
outside out
cell attached
dis/advantages of cell attached
measurement of ion channels in a specific region.
can change the voltage or add ligand. cell remains whole more physiological conditions inside the cell. each cell= 1 point on a dose response curve
dis/advantages of inside-out
inside= extracellular medium experimenter in control of the intracellular side of the membrane (bath). e.g cell activated by internal ligand. often forms a vesicle have to stop from forming vesicle through low ca2+ solution
dis/advantages of outside out
pippete= intracellular medium
can test the effect of external ligand.
often used for conc jumps
dis/advantages of whole cell
cell membrane ruptured electrode sees all channels contribution to membrane potential etc. cell contents washed out.
what is charge measured in?
Charge (Q) measured in Coulombs (C)
1 mole of monovalent ion= Faraday’s constant
Faradays constant=avogadro’s number x charge on ion (charge of +/- 1 = 1.6x10-19C)
what is current measured in?
Currrent (I) is measured in Amps
1 Ampere =flow of 1 C of charge in 1 second
I=dQ/dT
Ohms law
V=IR
I=V/R
Conductance
Conductance is the reciprocal of resistance
G=1/R so I=VG
summing up resistance
Series: add up resistance
Parallel: add up reciprocal of resistance
Capacitance
capacitance (C) in Farad (F)
1F= capacitance of an element that can store 1C of charge given 1V pd
How do you increase capacitance?
- Increase plate area (larger diameter axon)
- decrease interplate distance
- better conducting material between the two plates
summing up capacitance
in series: reciprocal adds up
in parallel: adds up
properties of ion channels
voltage dependence
activation
inactivation
timing of activation in respect to each other.
technical approaches to study ion channel
patch clamp
molecular cloning
crystallisation
main features of VGIC
water filled pore passive movement of ion down conc gradient selectivity filter gating properties to MP inactivation->refractory period
cause of selectivity filter
weak interaction between ion and charged AA
gating current
very small current
1/1000 of a normal current
recorded after all ionic currents are removed
confirmational change moves the charged portion of the channel- causes current ( ithink)
Cloning of Nav channel
isolate mRNA including ones encoding Na+ channel and make cDNA library
aa sequence of small region of Nav channel purified
oligonucleotide probe with sequence corresponding to aa sequence.
hybridise to cDNA library containing Na+ channel cDNA
isolate and sequence Na+ channel cDNA. deduce protein sequence
aa sequence of entire Na+ channel
tested in oocytes
hydorphobicity/philicity
ability of an aa to interact with water.
each aa has a specific value
how do you get DNA into eukaryotes
electroporation
ballistic method
DNA containing vesicle
calcium phosphatase
what changes the magnitude of an AP
conc gradient
motility of ions ( depends on size and interactions with water
temperature
what forms the resting membrane potential
selective permeability
unequal distribution
ion exchange pumps
(maintained by Na+/k+ ATPase)
Eion
membrane potential at which the concentration gradient is equal to the electrical gradient
potential difference
work needed to move 1 unit of +ve charge
give the cellular equivalent of a battery
concentration gradient across an ion
give the cellular equivalent of resistor/conductor
ion channel
give the cellular equivalent of capacitor
ability of a membrane to store charge
why does Vm decrease further from site of injection
more ion channels available- more leak
effect of lambda (membrane length constant) on signalling
High Rm, high lamda, PSP can travel further along axon before being lost
low Rm low lambda, lots of ion leakage
increase diameter of axon, increase lambda, potential spreads faster- less to impede current flow
lambda
effectively is the electrical conductivity of a neuron
what is an action potential
transient reversal of the membrane potential that sweeps along the membrane of a neuron
voltage clamp
xxxx
how can you separate ion currents
- modifying conc gradient of ion
- substituting critical ion with impermeable ion of same charge so electrochemical gradient is not changed
- selective ion blocker
difference between Na+ and K+ conductance
Na+ channels open faster and inactivate faster
K+ channels open more slowly and stay open longer
threshold potential
is the membrane voltage at which, enough Na+ channels are open to allow the overall flow of ions to be inwards
Refractory period generated by?
inactivation of Na+ channels and increased K+ conductance
AP propagation velocity depends on
axon diameter
passive membrane properties
effect of Ra and Cm on velocity
rate of passive spread inversely proportional to RaCm
because:
-large Ra, smaller current so will take longer to depolarise the adjacent membrane
-lage Cm, takes longer to charge the membrane therefore longer to depolarise.
how to speed up an AP
increase diameter of axon- reduces Ra as Ra=rax (pi x d^2/4)
C increases but linearly with radius.
Ra massively reduced Cm only slightly increased->RaCm decreased-speeds up AP
Myelination
C is inversely proportional to thicknes of membrane as capacitance increseases if conductors are closer together. saltatory conduction boosts the AP amplitude preventing it from dying out
active transport
movement of substances against their concentration gradient.
power sources for active transport
ATP hydrolysis
ion gradients
function of AT
establish electrochemical gradients pH regulation solute accumulation termination of synaptic transmission 2nd messenger regulation
an example of adhesion junctions
Cadherins
mechanical joining of cells
open lattice- allows fluid to penetrate deeper cell layers
Impermeable junctions
tight junction in vertebrates
prevents molecules and transporters on one side of cell from moving to the other
allows specific functionality of different sides
example of tight junctions
sealing of extracellular space in gut epithelium
active transporter on one side absorbs glucose
passive transporter on other side transport it to extracellular space so that glucose can not be lost back into the lumen of the gut
what are gap junctions made up of?
connexins- assemble into channels (either heteromeric or homomeric)
form half a channel- connexon
what do gap junctions do?
make cytoplasm continuous between cells can move cells up to 1000 molecular weight: -RNA -peptides -nucleotides -vitamins -sugars
where were they discovered
NMJ of cuttlefish
1958 Furshpon + potter
hyperpolarising current both from nerve terminal to muscle and muscle to nerve terminal
can demonstrate their existence using voltage clamp- as they form the potential of 2 cells come closer together
(loewenstein)
increasing size of dyes (pon et al 2007)
radioactive thymidine in healthy cell attatched to 1 mutant cell ko for thymadine kinase. radioactive thymidine found in both
where are they utilised?
heart to spread transmission between myocytes.- facilitates cotractile wave.
causes delay between atria and ventricles
smooth muscle in gut + uterus
function of glia
regulate excitability- regulates K+ and glu
signals to neurons
how many specialised processes do astrocytes have?
1- endfoot wraps around blood vessels
brings glucose into brain removes excess k+
BBB
what happens if glia do not remove excess K+
AP comes in K+ leaves cell
in a train of APs the extracellular K+ can rise by 10-20mM
this rise in K+ can cause change in membrane voltage- depolarisation- cell more excitable more likely to fire AP.
This can lead to epileptic discharge
waves of spreading depression
how does a wave of depression spread
K+ released from firing cell. makes neighbouring cells more excitable, makes them fire. wave of excitation causes Nav inactivation- depression in the brain moves a few mm at a time migraine
How do glia deal with high K+
take up K+ through channel- K+ taken up until there is no driving force
take up K+ through Na+/K+ exchanger
how much can a 10mM change in extracellular K+ change RMP
around 60mV
what is glial RMP
almost exactly Ek+ - not permeable to Na+
how does pump work
pump usually at 50% level. pump rate increases with K+ increase ( sigmoidal curve)
K+ pumped into glia with water- glia swell
Spatial buffering
if K+ rises on one side of the glial cell it will take it up and release some in other areas to maintain its own MP and to not raise K+ too much through out the cell.
K+ rise has to be all around the cell in order to depolarise the cell. depolarised cell will cause K+ release. may be relesed 100s of micrometers from where the rise is as glia are electrically connected by gap junctions form large networks
large amounts of K+ dumped by endfoot near the blood vessel and taken out of the brain
Proven by Newman 1984
K+ added externally at different area and extracellular measurements taken all round cell. largest change at endfoot- 90% of ion channels in end foot
what NT do glia cells help remove
Glutamate taken up into glia and broken down into glutamine via glutamate synthase either enters kreb cycle or taken up into presynaptic neuron by glutamine transporter
if not taken up quickly by glia can be neurotoxic
how does glutamate cause neurotoxicity
AMPA receptors activated, Ca2+ enters cell and activates Ca2+ dependent enzymes (phospholipases [breaks down membrane] and ribonucleases [breaks down genetic material])
AMPA- Na+ moves in depolarises cell, Cl- drawn in followed by water- cell swells and bursts
NMDA do not desensitise
what is needed for uptake of glut?
3 Na+ for 1 glutamate
causes depolarisation
how is blood flow increased to sites based on activity
Glia activated
astrocytes receive GABA B or mGlu stimulation->
Ca2+ rise-> PLA2 converts arachidonic acid into prostaglandin. released onto smooth muscle- causes relaxation through hyperpolarisation- increases blood flow
what is calcium used as a second messenger to do
NT release membrane excitability synaptic plasticity change in gene expression growth and differentiation neuronal death
