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