Excitability and Ion channels II Flashcards
Explain the activity occurring at the synapse - steps (9)
1) AP
2) Ca2+ influx (activated by CaV2 channels)
3) exocytosis - vesicles release neurotransmitters to act on
4) ligand gated ion channels or G protein couple receptors
5) neurotransmitter r on presynaptic cell = build up of neurotrans = excitation of post synap cell
6) act on either transport protein (back into pre-synap) or neurotransmitter degradation or uptake by glial cells
7) calphrin mediated endocytosis
8) large dense core granules
9) release from distant site - repeated simulation
How do we measure the activity? (2)
electrophysiology - by measuring capacitance (ability of the membrane to store charge) - specifically the changes in capacitance
- the flow of ions following activation
what does a large, wide spike suggest? (1)
multiple vesicles binding to the membrane + being released
what pattern is observed from large vesicles? (2)
ca2+ influx is always the triggering step
= neurotransmitter release is steeply dependent on the calcium concentration in pre-synap terminal
experiment w/ bath loaded w/ caged ca2+ + UV light (4)
- bound ca2+ released in response to to flashes of UV light
- controlling light = control ca2+ release
-non-linear relationship b/w ca2+ and it’s release - release of transmitters requires the binding of 5 ca2+ ions to a ca2+ sensing synaptic vesicle protein
measuring responses in the post-synaptic cell as a means of measuring neurotransmitter release experiment - 3 panels (6)
a) depolarising pulse
b) depolarising pulse + calcium (before stimulus)
c) calcium (after stimulus)
- only response was recorded was in B = sufficient mechanical mechanisms to release neurotransmitters
- no biolog response if ca2+ added after depolarisation
= must conclude ca2+ move from the extracellular space into intracellular to elicit a response
explain the SNARE Complex (both complexes)(3 +4)
exocytosis mediated by SNARE Complex
1)= tight interaction b/w synaptobrevin + synapsin + SNAP 25
2)Munc18 interacts w/SNARE Complex (essential for physical fusion)
1)2nd protein complex forms when Rab3A (vesical protein) binds to Munc13 + RIM
2) SNARE + Rab3A complexes = functionally linked because of an interaction b/w Synaxton + Munc13
3)Munc13 complex likely to inhibit the formation of the SNARE Complex = vesicle protein synaptotagmin serves as a ca2+ sensor for exocytosis + may also interact w/ RIM
4)synaptic vesicles also contain membrane transporters for reuptake -> synapsin is important for agnating the availability of vesicles in the reserve pole
Explain synapsin’s background + interaction (3)
-substrate for protein kinase A + Ca2+ calmoldulin binding protein kinase 1 = nerve terminal depolarised
= ca2+ enters synapsin = becomes phosphorylated by kinase + released from vesicle
=(mobilises reserve pool of vesicle transmitter for release)
name the 2 genetic deletion/application of synapsin antibodies (2)
-decrease in the no. of synaptic vesicles in the nerve terminal
-decrease in the ability to maintain a high rate of neurotransmitters release during repetitive stimulation
what do SNARE proteins do? (2)
catalyse the fusion of the vesicles w/plasma membrane because membrane bilayer is stable
(as it needs to overcome a large unfavourable Ea -> accompanied by family of fusion proteins (SNARES))
SNARE Complex summarised (4)
1) Synaptobrevin interacts w/2 plasma membrane target proteins
- synactin
-SNAP 25
= forms tight complex, brining the vesicle + pre-synaptic membranes in close opposition
2) Munc18 binds to SNARE complex
3) Ca2+ influx = triggers rapid fusion of vesicle + plasma membranes = snare complex now in plasma membrane
4)NSF + SNAP(new) proteins bind to complex = cause it to show association in ATP dependent reaction
CaV2 subfamily (2)
-all responsible for neurotransmitter release + dendritic ca2+ transients
CaV 2.1, 2.2, 2.3
Current types: N, P/Q, R
CaV2 subfamily in neurotransmitter release (3)
- Anchored in the active zone – nanodomain (CaV2.1 + 2.2)
- Regulated by voltage AND external agonists (e.g. dihydroprodrene = incr. channel openings) and antagonists (e.g. boscovity = inhibit neurotrans release)
- Also regulated by some GPCRs - specifically GPCR’s coupled to GQ pathway = reduces PIP2 in CaV2
GPCRs + CaV2 subfamily explained
- they inhib CaV2 channel via G-beta, gamma subunits
- coupled to GQ pathway = inhib by decreased PIP2 levels by modulation involving c terminal domain of CaV2 channel (incl. CDK5 phosphoryl mediated phosphorylation of a conserved serine of the CaV2.2
= increased channel opening probability = Ca2+ dependent inactivation (CDI) only present in Cav2.2 + 1) + Ca2+ dependence
= mediated by calmodulin binding to sites of proximal c terminal domain
Body signals in normal physiology- steps (3)
1) signal travels through body
2) gets to cell = activates receptor
3) physiological response (changes in morphology/shape of cell)
How does the cell translate an external signal into an internal signal? - 4 ways (4)
Direct: coupled directly to the drug
-Coupling to ion channels (ionotropic receptors)
Indirect:
- G protein coupled receptors (GPCRs) (metabotropic receptors/ 7 transmembrane r’s) -> coupled to a protein (50% of drug targets + responsible for most sensory signals in our body)
-Enzyme linked receptors -> coupling to enzyme
-Nuclear receptors (DNA transcription) -> bind to DNA = changes in DNA transcription
Name the key fast transmitters in the CNS (3)
Excitatory:
-Glutamate –> iGluRs (AMPA, Kainate, NMDAr) or mGluRs (Grp i/ii/iii)
Inhibitory:
-y-amino butyric acid (gaba) –> iGABARs or GABAbRs
-glycine –>iGlyRs
What are some other fast + slow transmitters (7)
Other fast synaptic transmitters:
ACh (nAChRs), 5HT (5HT3R), ATP (P2XRS)
Also many slow transmitters which often work by “volume transmission”-
DA, NA, 5HT, ACh, Neuropeptides, Histamine etc
Glutamate quick summary (2)
-Glutamate really is the major transmitter for synaptic communication
-Accounts for >90% of synaptic connections in the human brain
Many papers start “Glutamate is the major excitatory neurotransmitter in the vertebrate
nervous system….
AMPA Receptor Subunit Topology - learn diagram
Image
- Tetramer: for AMPA receptors GluA1-4 subunits in any combination
- 3 TM domains - M1, M3, M4 — redundant loop in M2
-*Q/R site – determines Ca2+ permeability of receptor
Properties of AMPA Receptors (5)
- Mediate majority of fast excitatory synaptic transmission (mainly postsynaptic localization) – AMPARs non-selective cation channels permeable to Na+ (goes in) and K+ (goes out) and in some cases Ca2+
- Like all ionotropic glutamate receptors comprised of four subunits = tetrameric receptor
- 4 different AMPA receptor subunits in mammals GluA1, GluA2, GluA3 and GluA4 these “mix and match” = produce subtly different receptors
- AMPA receptors containing GluA2 subunit have very low Ca2+ permeability
- due to mRNA editing – positively charged arginine (R) residue expressed instead of neutral glutamine (Q) in pore forming M2 region of GluA2
- = activation of all AMPA receptors = influx of Na+ but these receptors are only permeable to
Ca2+ only in the absence of any GluA2(R) subunits (and most AMPA receptors have GluA2(R))
Kainate Receptor Subunit Topology - (4)
- 3 TM domain + Redundant loop
-For kainate receptors GluK1-5 subunits – GluK4 or GluK5 expressed alone in a tetramer do not form functional receptors
*Q/R site – determines Ca2+permeability of GluK1 and K2
-GluK1 and GluK2 undergo RNA editing at a pore Q/R site and this is regulated in development
Impermeable to ca bless in early stages of dev (like AMPA)
NMDA Subunits - NR1 + NR2 (3)
-NMDA receptors are heterotetramers of two GluN1 (NR1) subunits and two GluN2 (NR2) subunits
-GluN1 (NR1) comes form a single gene but has 8 different splice forms…
– 4 different GluN2 genes which are differentially expressed in different brain areas and at different
ages
NR1 contains glycine binding site, NR2 contains glutamate binding site
3 important NMDA properties made up of subunits (3)
Three important properties:
* High Ca2+ permeability
* Mg2+ ions block channel at negative (i.e. resting) membrane potentials
* Glycine is a necessary co-agonist (in addition to glutamate)
NR1 contains glycine binding site, NR2 contains glutamate binding site
Mg2+ block of NMDA receptors is voltage-dependent (2)
for Mg2+ block of NMDAR :
- channel must be open i.e. glycine and glutamate must be bound to their binding sites on the NMDA receptor
Thus Ca2+ entry through NMDARs is dependent on pre- and postsynaptic elements being active at the same time i.e. the NMDAR is a coincidence detector.
How is the Mg2+ block of NMDAr channel removed (4)
As membrane potential is depolarized = the Mg2+ block of the NMDA receptor channel is removed
-AMPARs often present at same synapse as NMDARs
-Activation of AMPARs depolarises the
membrane sufficiently to remove Mg2+ block of NMDARs
-By voltage of ~ -35 mV the Mg2+ block of the
NMDA receptor channel is mostly removed i.e.
inward current through the NMDAR is voltage
dependent as well as transmitter gated
NMDAr synaptic physiology pattern vs AMPA (1)
Mediates a SLOWLY rising LONG LASTING excitatory
synaptic response via Na+ and Ca2+ entry through channel compared to AMPA.
NMDAr + Synaptic Plasticity (2)
The ability to change strength of synaptic
connections and consolidate new pathways in the CNS). NMDARs are involved in two processes - LTD + LTP
Via NMDAr Ca2+ influx
What does a current clamp measure? (1)
measures the membrane potential
What does a voltage clamp measure? (1)
records the current after being fixed at a specific membrane potential
What is Ohm’s law? (3)
V = I x R
- Voltage (V) due to separation of charge
Volts
-Current (I) represents the flow of charge
Ampere or Amps
-Resistance (R) oppose current flow Ohms
Current clamp (3)
Electrophysiological amplifiers are capable of stimulating the cell by injecting current.
Negative Current = causes hyperpolarisation of the membrane
Therefore, intracellular recordings of voltage are called ‘current clamp’
Voltage Clamp (1)
In voltage clamp, we are measuring what the amplifier is doing to keep the cell at Vclamp.
current vs voltage clamp in experiment - analogy of bank balance (3)
In current clamp VM will become more positive as it moves towards ENa
In voltage clamp VM cannot change (clamped). To
counteract the flow of +ve Na+ions into the cell, the
amplifier injects negative current
Analogy: In voltage clamp we are monitoring the ‘deposits’ and ‘withdrawals’ which are in the opposite direction to the ‘activity’
EPSP vs EPSC (2)
Why is the EPSC negative current?
The amplifier must inject negative current to maintain the cell at -80 mV to oppose the flow of
positively charged ions via the glutamate receptor
Extracellular recording (3)
Place electrode outside and add current to axon = depolarised all CELLS and not one cell = measure activity of one cell (easier)
As positive ions move into the cell, the extracellular
space becomes more negative
We call this a field potential or a fEPSP
electrophys recording summary (4)
- Intracellular electrophysiology used to study electrical activity in single cells
-Extracellular electrophysiology used to study electrical activity from groups of cells in one anatomical location
-Current clamp recording can be used to monitor the effects of manipulations (eg drugs) on
membrane potentials
-Voltage clamp can be used to study the underlying biophysical properties of channels in the
membrane and the effects of manipulations (eg drugs) on these
What is always the triggering step? (1)
The calcium influx
4 methods of eliciting a response in detail (4)
Ligand gated ion channels: (ms)
Binding of r = confirmation change = influx of Ions -> hyperpolarisation/depolarisation -> cellular effects
GPCRs: (s)
Bind to protein -> either phosphorylation of other proteins or kinases coupled with ion channels
Kinase/enzyme linked r: (hrs)
Receptor linked to it -> protein phosphorylation -> gene transcription -> protein syn
Nuclear r: (hrs)
Hypophilic: elicit response at targets
- mediate gene synthesis = response
Glutamergic synaptic transmission + AMPA blockage (2)
Mechanism of action: ionotropic
ESPC can be almost completely blocked by selective AMPA r antag eg Telampanel
