Neuronal signalling Flashcards
Electrical properties of membranes can be measured using
- a black-lipid bilayer
- membrane acts as a capacitor
Capacitance
C = Q/V
How to measure the conductance of purified ion channels or vesicle preparations?
reconstituted into black-lipid bilayers
Channels act as conducters
allowing ion flow across the membrane
conductance (g)
- reciprocal of resistance
- 1/R
- unit = Siemens, S
Give the circuit analogue view of a channel
- pump
- membrane capacitance
- channel conductance
The average current-voltage relationship of open channels follows
Ohm’s Law
The convention for ion currents is that
positive charge flowing out of the cytoplasm is a positive current
Conductance is measured graphically as
the gradient (m)
A concentration gradient of an ion establishes
a diffusion potential
Give the The Nernst Equilibrium potential for K+:
2.3 (𝑅𝑇/𝑧𝐹) 𝑙𝑜𝑔^10 ([𝐾^+ ]𝑜𝑢𝑡/[𝐾^+ ]𝑖𝑛)
The equilibrium potential shifts
the average current-voltage relationship along the axis
Give the equilibrium potential for chloride
-70mV
Give the equilibrium potential for potassium
-90mV
Give the equilibrium potential for sodium
+60mV
Give the equilibrium potential for calcium
+130mV
The activity of single ion channels can be measured using
patch-clamp techniques
Describe the effect of patch clamp configuration
- different patch-clamp configurations give access to either side of the membrane
- pulling without suction = inside-out patch
- pulling with gentle suction = outside-out patch
Time-averaged current depends on
frequency and duration of channel opening events
Membrane potential affects
the open probability of the channel: voltage-gating
Describe the KcsA channel from Streptomyces lividans
- four subunits, each with 2 membrane helices (outer and inner) and a pore loop
- surround a central pore cavity
- turret
- K+ ion and crystal water
Ion selectivity is determined by
- ionic radius
- hydration energy
Ionic radius is measured in
angstrom
Hydration energy is measured in
kcalmol-1
Describe Li+ and Na+ trying to pass through a K+ channel
- small enough
- only if sufficient energy is recouped from interactions with pore residues to overcome the hydration energy required to strip off the associated water molecules
Describe the selectivity filter in KcsA
created by juxtaposition of the four P-loop chains
Describe the S4 membrane spanning domain
- inward Kirs have four subunits
- each subunit has six MSDs
- Na+ and Ca2+ channels have four domains, each with six-membrane spanning alpha-helices, including four copies of the S4 voltage sensor
- positively charged arginine or lysine amino acids every third residue
- helix moves in response to the membrane potential; affects the conformation of the channel protein opening (or closing) the pore
Kirs
rectifying K+-channels
MSDs
membrane spanning domains
Voltage gating is associated with
movement of charges within the protein
Channels may show
time-dependent kinetics
Describe delayed-rectifier channels
- delay before full conductance due to recruitment of channels from an inactive form
- time-dependent inactivation
- e.g. K+
Describe a voltage-gated Na+ channel
- activates rapidly
- followed by rapid decrease in conductance due to channel inactivation
Describe time-dependent inactivation
via pore occlusion
Describe chemical coupling via synapses
- pre-synaptic axon
- synaptic cleft
- post-synaptic dendritic spine
Describe the initiation of an action potential via nAChR
- acetylcholine released from the pre-synaptic membrane activates the receptor
- a low conductance K+ ‘leak’ channel sets the resting membrane potential close to Ek
- although the ACh receptor will conduct both Na+ and K+, only Na+ moves initially as Na+ is far from equilibrium
- influx of Na+ causes membrane depolarisation
nAChR
nicotinic acetylcholine receptor
Describe a channel conductance graph
- foot
- rising phase
- falling phase
- refractory period
List the types of synapse
- neuromuscular junctions
- synapses with another neurone
- neuroglandular synapses
Describe neuromuscular junctions
- neurone
- collateral branch
- telodendria
- synaptic terminals
- neuromuscular junctions
- skeletal muscle fibres
Describe synapses with another neurone
- dendrites
- collateral branch
- synapses with another neurone
- axolemma
- neurone 2
Describe neuroglandular synapses
gland cells
The motor end-plate is
a modified synapse
Describe the sarcomere
- fibrils
- outer vesicles of SR
- PM
- longitudinal tubules of SR
- transverse tubule
- Z line
- I band
- thin filament
- H zone
- M line (bare zone)
- overlap region
- A band
- triad
SR
sarcoplasmic reticulum
How can sarcomeres be visualised?
X-ray crystallography
Describe EPSPs
- increase the likelihood of a post-synaptic action potential occurring
- typically mediated by glutamate receptors
- allow influx of Na+ or Ca2+
EPSPs
Excitatory Post-Synaptic Potentials
Describe InhibitoryPSPs
- decrease the likelihood of a post-synaptic action potential
- typically mediated by GABA receptors
- allow influx of Cl-
Describe optogenetics
- bringing neurons under experimental control in intact organisms
- modulate targeted neurone activity using light
List some light-activated channels and pumps
- blue-light dependent depolarisation using ChR2
- yellow-light hyperpolarisation using Halorhodopsin chloride pumping
ChR2
- Channel Rhodopsin 2
- K+ out
- Na+ + Ca2+ in
- controls neuronal activity
- visualised in nematode mobility
Six steps to optogenetics
- piece together genetic construct of promotor (to drive expression) and gene encoding opsin
- insert construct into virus
- inject virus into animal brains (opsin expressed in targeted neurones)
- insert optrode
- laser light of specific wavelength opens membrane ion channel in neurones (sodium influx)
- record electrophysiological and behavioural results
opsin
light-sensitive ion channel
oprtode
fibre-optic cable plus electrode
Describe optogenetics and aggression
pptogenetic stimulation of neurons in VMHvl
VMHvl
ventromedial hypothalamus, ventrolateral subdivision
What happens if you touch and optogenetic nematode’s body muscles?
forward locomotion
What happens if you touch and optogenetic nematode’s neck muscles?
head movements
