Jon Turner week 7-9 Flashcards
1
Q
6 nov wut?
A
wutwut wtf jon
2
Q
what is the difference between pores and channels?
A
pores are always open, channels are gated.
3
Q
describe the structure of channels.
A
inner vestibule - between selectivity filter and gate
outer vestibule - outside selectivity filter.
gate
selectivity filter in the middle.
4
Q
what are ion channels all made of?
A
proteins, polypeptide chains.
5
Q
describe the selectivity filter on a Na channel.
A
Short, wide pore - inner ring of DEKA (Asp, Glu, Lys, Ala) side chains - net charge: -1
Outer ring of EEMD (Glu, Glu, Met, Asp) - helps stabilizes waiting ions - net charge: -3
6
Q
describe the selectivity filter on a Ca channel.
A
Again a short, wide pore containing a ring of EEEE (Glu) side chains - net charge: -4
Outer ring of DSED (Asp, Ser, Glu, Asp) - again helps stabilizes waiting ions - net charge: -3
7
Q
describe the selectivity filter on a K channel.
A
Tube of 4 x TVGYG (Thr, Val, Gly, Tyr, Gly) - all relatively neutral residues with no side chain present in the narrow pore - weak charge
8
Q
what are the states of a channel?
A
open/closed - open state allows ion transfer.
transition - between the states this is referred to as gating.
9
Q
what is gating?
A
the transition between open and closed/vice versa.
10
Q
how is generalized gating achieved?
A
Twisting, tilting or bending of subunits and trans-membrane spanning α-helices (much of α-subunit around the pore region).
11
Q
describe mechanical distortion
A
Opened by distortion of the plasma membrane (mechano-sensitive).
↑ opening induced either directly or through mechanical linkage to the cytoskeleton.
Sensory transduction in sense organs such as cochlea and skin by TRP channels. Also some “leakage” K+ and Cl- channels
12
Q
difference between activation, deactivation and inactivation?
A
activation and deactivation are open/closed.
inactivation is permanently shut.
deinactivation is undoing of this.
13
Q
describe inactivation/deinactivation.
A
localized:
Either a region in pore wall or close to it alters conformation physically occluding the pore
Or the selectivity filter changes conformation, reducing ion transfer.
Involves a relatively short amino acid sequence.
OR particle induced:
A free intracellular region of the channel protein that plugs the pore; a.k.a. “ball and chain” gating.
Involves a relatively long amino acid sequence or subunit.
14
Q
origins of the resting K membrane permeability?
A
Julius Bernstein 1902, Halle, Germany (long before anyone knew ion channel existed! Had to wait until late 1970’s, early 1980’s)
Proposed the “membrane hypothesis”
Membrane was ion-selective for K+ and so K+ was the most likely ion to be responsible for RMP as:
- extracellular K+ concentration was known to be low
- indirect evidence pointed to RMP being negative
1st person to apply the Nernst equation to excitable cells:
not proved until intracellular recordings were invented.
15
Q
how did hodgkin and horowicz test the membrane hypothesis?
A
Modified EK+ by changing [K+]o
Intracellular recording from a frog muscle fibre @18°C
Record RMP at varying [K+]o
Should obey the Nernst eq.
Result:
>10mM [K+] data fitted by Nernst eq.
K+ channels contribute background ion permeability for RMP >10mM
16
Q
how do ion channels vary in ion selectivity?
A
sign and density
+ -
2+ +
17
Q
is selectivity relative or absolute?
A
relative.
K channels allow Na to pass, if exta/intracellular K was removed they would become Na channels.
18
Q
what is an equivalent circuit?
A
An equivalent circuits is a theoretic circuit that retains the properties of whatever is being modelled in its simplest electrical form
Associates membrane currents with specific ion channels with Eions and their conductances as well as the membrane’s capacitive properties
19
Q
describe a battery or ion pump
A
Represents the electrochemical ion gradient - a source of electrical energy (equivalent to a power source or battery)
Batteries generates a voltage known as electromotive force (emf)
For each set of ion channels:
emfion = Eion
Force that drives Iion is the driving force or voltage relative to Eion i.e. the difference between the measured transmembrane voltage Vm and Eion:
Vm - Eion
larger driving force = larger current
Units: Volt (V)
DRAW ALL DESE OUT
20
Q
describe conductance and resistance
A
Represents the ion channel permeability - a measure of how readily a current will pass through a material (G).
therefore opposite of resistance (R), a measure of how much the material opposes the flow of current
Assigned Units: Siemens (S) or ohm-1 (Ω-1)
Represented as a variable (left) image rather that a constant (right) image
21
Q
what is capacitance?
A
Capacitors are devices that store charge
Capacitance is a measure of that ability
Capacitance (C) = stored charge(Q) / voltage(V)
Two conductors separated by a non-conductive material -dielectric/insulator
LIKE A MEMBRANE GEDDIT
Intracellular and extracellular fluid are the conductors, the membrane is the dielectric that maintains charge separation
Units: Farad (F)
22
Q
how does potential difference change the stored charge if there is capacitance in a system?
A
increase -> increase
decrease -> decrease
23
Q
what is a capacitance current?
A
current required to charge/discharge areas adjacent to the membrane
24
Q
what determines total capacitance?
A
surface area of a cell membrane.
bigger = bigger
25
what is kirchhoffs current law?
Total charge flowing into a point or node must be the same as total charge leaving the point or node.
For a steady state potential this is okay, Ic = 0
26
what is the simplified equivalent circuit model?
Em Gm in out
| DRAW IT and equations
27
at a steady state what does Igm =?
0
28
what is net conductance?
Gm
29
what is net conductance associated current?
IGm
30
at a steady state what does Vm =?
Vm = Em
31
describe passive responses from the membrane potential.
injecting a current alters the membrane potential.
has a slightly weaker response, check diagram.
current flows through the membrane.
During response, Vm no longer = RMP
32
what is Ic?
capacitive current
33
what is IGnet
net ion channel current
34
dynamics over time wut
wutwutinthebutt
35
what is the time constant?
time taken to reach change in voltage (ΔVm) = 63%
pi = C x R
36
what is Iinj?
Ic + IGm
37
capacitance equation?
C = Q/V
38
relationship between rate of membrane potential change to membrane capcitance?
the rate of membrane potential change (for any given current) is inversely proportional to the membrane capacitance.
the greater the membrane capacitance the slower the rate of change of membrane potential (for any given current).
39
impact of capacitance slide?
???
40
relationship between conductance and time constant?
↓ conductance (G) → ↑ time constant () and vice versa
41
how do voltage responses differ to membrane currents?
Voltage responses are going to lag behind any membrane currents!
42
how does neuromodulation act?
affects the resting conductance.
ie shutting K channels would increase excitability.
43
describe short or long stimuli.
Brief/fast current stimuli need to be much larger than long/slow ones in order for the associated voltage change to reach threshold.
44
What is a voltage clamp?
amplifier monitors membrane voltage and then estimates and injects current necessary to maintain voltage level set by user called Vcommand using “a negative feedback system”
Purpose to measure current flow across membrane and so determine current flow and conductance of ion channels responsible for the current.
Current injected to keep voltage constant is equal but of opposite polarity to trans-membrane current being generated causing membrane potential to change any point in time.
45
what does E/V mean?
electromotive force/voltage
46
what does Q mean?
(quantity of) charge
47
what does I mean?
current Intensity
48
what is G/gamma?
God only knows!!!??!!
conductance.
49
What is Erev?
Erev is the potential at which macroscopic current flow reverses direction.
So typically, if channels highly ion selective: Erev = Eion
50
what is macroscopic current?
whole cell conductance x driving force
I = G (Vm - Erev)
51
benefits of looking at the macroscopic current?
many channels contribute to observed behaviour.
reliable
Good indication of how cell behaviour would be affected over time.
52
pioneers of the voltage clamp?
alan hodgkin and andrew huxley
First to describe and characterize Na+ and K+ currents in the membrane of the squid giant axon in 1950’s using an early version of the voltage clamp technique.
Hodgkin & Huxley had no pharmacological tools - replaced an active ion i.e. Na+ with a non-permeating one e.g. choline+
decrease conc of Na, less depolarisation occured on the AP.
53
describe the squid giant axon.
Squid giant axon - large diameter fibres found in stellar nerve up to 1mm diameter usually 500-600μm.
Coordinate water ejection from mantle cavity during swimming and escape response.
Unmyelinated axons - conduction velocity - 25m/s
depolarise from -20 --> -80 for action potential
54
H and H basic VC circuit?
1: A voltage protocol to be used is set by voltage command Vcom
2: The first stage of the VC amplifier measures membrane potential Vm
3: The second stage of the VC amplifier compares Vm to the predefined Vcom protocol
4: This second stage then adjusts the current flowing to a second intracellular electrode to bring Vm equal to Vcom as quickly as possible - to nullify the current causing any difference
5: If this feedback system is fast enough then the current will match that flowing at the membrane in terms of amplitude and kinetics
?????????
55
describe hyperpolarisation and depolarisation of a giant squid axon.
hyper:
Very small inward current (about -30μA/cm2) due membrane leakage conductance (K+)
depol:
Transient inward followed by a sustained outward current
transient capacitive current rapid spike at the beginning.
56
effect on giant axon by blocking Na and K?
K:
Reversal between +30 and +60mV
Na:
lack of inactivation on +30/60
slower onset to activation
diagrams
57
H&H OG findings?
Fast Na+ current:
Activation range -50 to 20mV
Rapid and complete inactivation
Na+ current reversal close
to ENa+ @ +40 to +50mV
“Delayed Rectifier” K+ current:
Activation range -50 to 20mV
Slow activation, non-inactivating
K+ current reversal below activation threshold close to EK+ @ -90 to -80mV
58
describe single channel recordings.
Microscopic
timing is not precise, length they are open can vary, they are unreliable
Usually only one or a few channels of the same type
Current flow is relatively small
59
advantages of macroscopic?
mean of lots of microscopic gives a more accurate depiction.
60
what is the equation for total membrane current?
I = N x Fo x i
Fo = fraction of channels open at any time
61
what is the equation for total membrane conductance?
G = N x Fo x γ
62
what happens when a system is ohmic?
chord = slope
as both measures pass through or can be extrapolated back to Erev.
Lienar
63
graph things idk wut
wuwtu
64
what are some atypical excitable tissues?
Heart
Sino-atrial node - pacemaker potential
Cardiac myocytes - ventricular action potential
Pancreas
Insulin-secreting β-cells - sensing glucose
Sense organs such as the retina
Graded responses in photoreceptors
65
describe the cardiac conduction system.
Different regions all electrically active.
Need to coordinate timing of atrial and ventricular contractions for efficient movement of blood.
Signals initiated by spontaneously active region sino-atrial (SA) node on right atrium → ventricles.
Linked by gap junctions - small pores that link the cell membranes of one cell to the next, allow current to flow through.
66
how do cardiac potentials change depending on the location?
Electrical activity varies depending on location and cells generating activity:
Sino-atrial (SA) and atrio-ventricular (AV) nodes - slow upstroke, little plateau
Atrial and ventricular myocytes plus Purkinje fibres - fast upstroke, long plateau.
graph makes sense
67
describe the sino atrial node potential structure.
Phase 0: upstroke
Ca2+ current (ICa) slow depolarization - L-type Cav1.2
Phase 3: repolarization
K+ current (IK) repolarization - a combination of Kir (Kir3.1/3.4) and Kr (rapid delayed rectifier or hERG - human Ether-à-go-go related gene channels, Kv11.1)
Phase 4: pacemaker phase
If (f for “funny”) - pacemaker current (during diastole)
IT (T for “transient”) - subthreshold Ca2+ current Cav3.1
Slow rising ramp-like potential that initiates the next upstroke
Same in the atrial-ventricular (AV) node
graph it
68
what is If?
Ifunny - not activated by deplarisation, only hyperpolarisation with a threshold between -50 to -40 mV.
Eion between -20 to -10 mV due to mixed permeability to Na+ and K+
69
what underlies If?
HCNs - hyperpolarization activated cyclic nucleotide gated channels.
ie AMP/GMP (ATP/GTP????)
intracellular domain has the cyclic nucleotide binding site, determines the voltage dependence of the channel.
shift voltage dependence +/-
CNBD - cyclic nucleotide binding domain (cAMP/cGMP) - shifts voltage-dependence of activation to a more positive (depolarized) level.
70
describe the autonomic modulation of the If in the heart.
Parasympathetic input - vagus nerve (Xth cranial nerve) ↓ heart rate (bradycardia)
Neurotransmitter: Acetylcholine ↓ If, ↑Kir, slows pacemaker firing rate (green) via M2 muscarinic receptors - Gi →↓cAMP
Sympathetic input - T (thoracic)1-4 spinal nerves ↑ heart rate (tachycardia)
Neurotransmitter: Noradrenaline (NA) ↑ If, speeds up pacemaker firing rate.
Mimicked by Iso - isoprenaline βA-R agonist, (red) via β1 adrenergic receptors - Gs → ↑ cAMP
71
what does Gi do?
M2 muscarinic receptors.
| inhibits adenylate cyclase and reduces intracellular cAMP levels.
72
what does Gs
B1 adrenergic receptors.
| stimulates adenylate cyclase and increases intracellular cAMP levels
73
describe the ventricular myocyte action potential.
Phase 0: upstroke
Activation of Fast INa+ NaV1.5
Phase 1: initial rapid repolarization
Inactivation of Fast INa+ NaV1.5
Transient IK+ KV4.2/4.3 (Ito)
Phase 2: plateau phase
ICa2+ L-type CaV1.2,
IK+ slow delayed rectifier KV7.1 (Ks)
Phase 3: terminal repolarization
IK+ slow delayed rectifier KV7.1 (Ks)
IK+ rapid delayed rectifier KV11.1 (Kr - hERG)
IK+ inward rectifier Kir2.1/2.2/2.3
Phase 4: resting (flat)
IK+ inward rectifier Kir2.1/2.2/2.3
graph
74
describe hERG channels
hERG - human homolog of Drosophila Ether-à-go-go channels.
Ether-à-go-go a mutant fruit fly:
when anaesthetised with ether their legs shake like the go-go dancers at the famous LA nightclub!
hERG channels currents unusual:
typical activation >-40mV
partial inactivation when membrane potential depolarized >0mV
rapid reversal on hyperpolarization → apparent increase in current/conductance
“paradoxical resurgent current”
75
what is long QT syndrome?
prolonged plateau
Definition: abnormally long interval between onset of excitation and contraction of the ventricles and their subsequent relaxation
i.e. interval between depolarization (0) and terminal repolarization (3)
caused by some drugs e.g. methadone, erythromycin, haloperidol
drugs need to be tested for hERG blocking action
caused by some gene mutations (need to know??)
76
how does a long QT connect to ventrical fibrillation?
prolonged action potential leads to early after depolarization.
this can lead to a run of spontaneous activity.
77
exocrine and endocrine?
Exocrine – secretion of digestive enzymes by pancreatic acini
Endocrine – secretion of hormones associated with metabolism by Islets of Langerhans
78
makeup of cells in the pancreas?
insulin secreting β(B)-cells (65-90%)
glucagon-releasing α(A)-cells (15-20%),
somatostatin-producing δ(D)-cells (3-10%)
pancreatic polypeptide-containing F(PP)-cells (1%)
79
how do B cells respond to different levels of glucose?
low glucose levels: hyperpolarised
high glucose levels:
rhythmic depolarizations and high threshold spiking.
leads to insulin release.
80
describe the mechanism by which B cells respond to glucose.
glucose enters cell, becomes phosphorylated, mitochondria process it via krebs cycle and produce ATP.
ATP sensitive K channel sensor for levels of ATP relative to ADP within the B cells.
As ATP levels rise functionality of K channel decreases, depolarisation due to more intracellular K.
voltage dependent Ca channels trigger, Ca enters and triggers the insulin release.
In jons words:
Glucose transported by type 2 glucose transporters (GLUT2) and phosphorylated, trapping glucose inside.
Eventually, ATP is produced in the mitochondria.
The increase in ATP:ADP ratio leads to closing of ATP-sensitive K+ channels. These contribute to RMP -70mV.
Membrane potential depolarizes under the influence of other channels contributing to resting potential.
Voltage-gated calcium channels open, allowing calcium ions (Ca2+) to enter.
6. The increase in intracellular calcium concentration triggers the secretion of insulin via exocytosis from secretory vesicles.
81
describe SUR/Kir6.2 channels
underpin insulin release.
Four inward rectifier K+ channel subunits - Kir6.2
associated with four regulatory sulfonylurea receptor (SUR) subunits - SUR1, an ABC (ATP-binding cassette) protein
diagram.
4 kir6.2 surrounded by 4 SUR1.
Binding of ADP to SUR1 → opening, binding of ATP to Kir6.2 → closing
sensitive to ratio of [ATP] : [ADP]
82
define type II diabetes mellitus.
Definition: hyperglycemia due to low insulin production by -cells, or insulin receptive cells showing insulin resistance
83
how can type II be treated?
Phamacology: first line of defence to reduce gluconeogenesis in the liver (the biguanide - metformin)
If this is insufficient, then sufonylureas used to increase insulin release. First line of defence until 1990’s
Sulfonylureas target the SUR1 subunit
e.g. tolbutamide, glibenclamide
bind to ATP-sensitive K+ channel
more in a closed state
closed state → ↑ insulin release.
84
What happens if SUR/Kir6.2 have a loss of function mutation?
“Loss of function” mutations → depolarization → ↑ insulin secretion
highly sensitive to ATP/ADP ratio
Congenital hyperinsulinism, overproduce insulin.
e.g. mutation of Kir 6.2 - persistent hyperinsulinemic hypoglycemia of infancy (PHHI)
85
What happens if SUR/Kir6.2 have a gain of function mutation?
“Gain of function” mutations → less sensitive to high ATP/ADP ratio → hyperpolarization → ↓ insulin secretion
Neonatal diabetes; Predisposition Type 2 diabetes
86
how do photoreceptors respond to light?
“Resting” membrane potential depends on light intensity
↑ light → hyperpolarization
↓ light → depolarization
If the dogma is that depolarization equates to increased responsiveness then photoreceptors respond to the dark!
87
describe the mechanism of photoreceptors.
cGMP cyclic nucleotide gated (CNG) channel
Non-selective mixed cation channel Erev ≈ 0mV
Open when cGMP binds and closed when cGMP dissociates (unbinds!)
Intracellular ligand-gated!
In dark, cGMP levels high → CNGs open → depolarization → 0mV
In light, increased PDE ↓ cGMP → CNGs close → hyperpolarization → EK+
?????
88
chemical v electrical synpase
chemical:
- Molecules stored in vesicles
- Molecules diffuse across a “gap” between cell membranes
- Signalling: relatively slow (0.5msec)
- Unidirectional
- Majority of transmission in the nervous system
electrical:
- “Holes” in adjoining cell membranes: linked by channels - gap junctions
- Signalling: very fast - near instantaneous
- Bidirectional
- Relatively rare in nervous system
- Direct electrical coupling between cells
- ions, second messengers, metabolites
89
function of chemical synapses?
Allow modification of nervous system function:
Neural computation - integration of many input.
Exhibit plasticity - development, learning and memory.
Act as targets for drug action - neurotransmitter synthesis, release, receptors, uptake, degradation.
90
how is a neurotransmitter defined?
- Synthesised and stored in the pre-synaptic neurone.
- Released upon stimulation of that neurone in a depolarization and Ca2+ dependent manner.
- Compound must reproduce physiological effects when applied to postsynaptic neurone.
- Located in the appropriate region at levels sufficient to evoke physiological responses.
- Transmitter recognition & signal transduction mechanisms (receptors etc.) associated with that postsynaptic neurone.
- Transmitter removal mechanisms.
91
types of neurotransmitters?
small molecules:
amino acids, amines, purines.
peptides
92
examples of amino acids?
glycine
GABA
glutamate
93
examples of amines?
noradrenaline
dopamine
serotonin
acetylcholine
94
examples of purines?
ATP
| adenosine
95
examples of peptides?
endorphines
| somatostatin
96
what is dales principle?
neurones release just one transmitter at all its synapses.
not true.
This is challenged by the co-existence and release of small transmitters + peptides by interneurones.
97
types of vesicles?
small synaptic vesicles (SSVs)
large dense cored vesicles (LDCVs)
differences on table lecture 15
98
describe SSVs
located in synapse active zones
contain small neurotransmitters
200um Ca for release
single APs
biogenesis:
Constitutive - local vesicle recycling, transmitter synthesis and uptake
99
describe LDCVs
location is non specific, diffuse all over cell.
contain peptides (sometimes noradrenaline)
5-10um Ca for release.
repetitive activity.
Biogenesis:
Regulated - ER-derived vesicles (no recycling), transmitter production and processing under direct genomic control
100
Heuser and reese vesicle experiment?
Heuser and Reese - “Slam freezing” rapidly cooled on a metal block after electrical stimulation of frog neuromuscular junction
Freeze fracture electron microscopy to visualize the presynaptic membrane
Sections of presynaptic membrane take at different times after a single nerve impulse
```
found that:
Vesicles 40-50nm diameter,
Clear centres,
Spherical,
Could contain 1000’s of neurotransmitter molecules.
```
Problem: activity leads to a massive increase in membrane surface area
Answer: Vesicle recycling
101
classic vesicle cycle (brief)
```
docking
priming
fusion/exocytosis
endocytosis
recycling
```
102
describe docking in the vesicle cycle
Synaptic vesicles only dock at the active zone, active zones differ between neurones
This is adjacent to signal transduction machinery of postsynaptic membrane leading to enhanced speed and precision of signaling.
103
describe priming in the vesicle cycle.
Synaptic vesicle “maturation” process
Vesicles made competent to release transmitter (in response to Ca2+)
Requires ATP
Conformational change in proteins that drive release
104
describe exocytosis in the vesicle cycle.
Fusion of synaptic vesicle and presynaptic terminal membrane
Requires Ca2+
Involves Ca2+ “sensor” protein
Fusion induces exocytosis - contents discharged (diffusion)
Takes about 1msec
105
describe endocytosis in the vesicle cycle.
Synaptic vesicle membrane is recycled by endocytosis
Involves cytoskeletal protein lattice formation (from clathryn monomers) to help pinch off membrane
Takes about 5 secs.
membrane becomes coated in a protein called clathryn
??
106
describe recycling in the vesicle cycle.
Mechanism to conserve synaptic vesicle membrane via endosome
decoated of clathryn.
Vesicles refill with transmitter (ATP-dependent again - concentrating neurotransmitter)
107
what is the alternative model of the vesicle cycle?
kiss and run.
Full vesicle fusion may not be required to release transmitter:
SSVs recycle intact
Not via endosomes
Neurotransmitter leaks out of small fusion pores on the membrane.
108
comparison of two vesicle cycles.
Kiss and Run Mechanism:
Fast
Low capacity - only a few vesicles at a time
Favoured at low frequency stimulation
Classical Mechanism:
Slow
High capacity - many vesicles at a time
Favoured at high frequency stimulation
109
what is v-SNARE?
Synaptobrevin (a.k.a. VAMP)
18kDa
Single transmembrane spanning
part of the protein is in the cytosol.
110
what is t-SNARE?
Syntaxin
35kDa.
Single transmembrane spanning.
```
SNAP-25
Synaptosomal-associated protein 25.
25kDa.
Anchored to membrane by acyl chain.
functional part of protein in the cytosol.
```
111
describe the structure of the snare proteins.
Synaptobrevin/syntaxin/SNAP-25 make a 1:1:1 trimeric complex.
a coiled coil quaternary structure - α-helices (two from SNAP25 green/blue)
112
the role of snare proteins in docking?
helps attach the vesicle to the membrane
113
the role of snare proteins in priming?
change in conformation of snare proteins, become loosely associated.
Munc 18 interacts and causes tighter bonding "zippering".
ATP dependent.
fusion can now occur.
“zippering” - formation of the SNARE-pins
114
role of snare proteins in fusion?
The Ca2+ sensor - synaptotagmin found on vesicles
Synaptotagmin binds to SNARE-pins in absence of Ca2+ (priming)
Synaptotagmin binds to phospholipids in presence of Ca2+
Ca2+ may cause synaptotagmin to pull vesicle into membrane (fusion)
115
role of snare proteins in recycling?
Association of SNAREs drives docking, priming and fusion
BUT SNAREs must dissociate to allow:
- Internalisation of empty vesicles
- Re-docking of another vesicle
This involves NSF (N-Ethylmaleimide-sensitive factor) another ATPase.
NSF binds to SNARE-pin complex to facilitate dissociation.
116
describe botulinum toxins
Made by the anaerobic bacterium Clostridium botulinum.
Exposure either via ingestion of infected food or by infiltration of a wound.
One of the most deadly toxins known - type A most lethal (1ng/kg)
Death by respiratory failure to paralysis of respiratory muscles
The disease produced by one or more of these toxins is known as "botulism".
Affects peripheral nervous system: flaccid paralysis of skeletal muscle and autonomic nervous system dysfunction - BUT does not cross BBB
Clinical/cosmetic uses: convergent squint (strabismus), excessive sweating, the effects of aging!
Potency so great - no one has yet quantified the minimum concentration, or minimum number of molecules, needed to disrupt function.
Use in chemical weapons or acts of terrorism! NEEDS STRICT REGULATION!!
Botulinum toxins are a group of seven neurotoxins (designated A-G)
Made as single polypeptide -1296 amino acids (Mr 150,000)
Nicked to give two chains: a 50kDa chain disulfide linked to 100kDa chain
117
structure of botulinum toxins?
Botulinum toxins are a group of seven neurotoxins (designated A-G)
Made as single polypeptide -1296 amino acids (Mr 150,000)
Nicked to give two chains: a 50kDa chain disulfide linked to 100kDa chain
3 functional domains:
Catalytic domain: Zinc protease
Translocation domain: allows it to cross lipid membrane
Receptor binding domain: cholinergic specificity/endocytosis
diagram it
118
how does botulinum act?
targets t/v snares.
protease.
disrupts them, prevents release of NT.
119
distribution of neurones in the brain?
Human CNS - 86 x 109 (Herculano-Houzel)
Glutamate ↑70% 60 x 109
GABA ↑30% 26 x 109
“Neuromodulators”
120
describe excitatory NTs
glutamate 5C
ubiquitous (“everywhere”)
cerebral cortex → spinal cord
60-70% of all synapses
121
describe inhibitory NTs
γ-Aminobutyric acid (GABA) 4C - cerebral cortex → brain stem
Glycine 2C - brain stem → spinal cord
20-30% of all synapses
122
types of synapses in CNS?
```
gray type I
- asymmetric.
- “Excitatory” - associated with
L-glutamatergic synapse markers
- spherical vesicles
```
```
gray type II
- symmetric.
- “Inhibitory” - associated with GABAergic and glycinergic
synapse markers.
- flattened vesicles
```
123
reversal potential?
no response
| ??
124
types of NT receptors?
ionotropic:
receptor-ionophore complex from ligand binding
fast
metabotropic:
GPCR
membrane bound proteins linked to an intracellular G protein, hydrolyses GDP to become activated. Activates a 2nd messenger.
slow
125
what are the glutamate receptors?
AMPA
Kainate
both homomeric or heteromeric.
antagonist - Quinoxaline-2,3-dione - NBQX
NMDA
heteromeric only - dual agonism with glycine.
NMDAR2 binds glutamate
NMDAR1 binds glycine
antagonist - 2-amino-5-phosphonopentanoate (D-AP5)
all ionotropic
Non-selective mixed cation channels.
Eion 0mV
tetramers.
126
voltage dependence of fast excitatory neurotransmission?
at -80mv resting
AMPA does most, little NMDA
at -40mv resting
AMPA less effective, more NMDA
127
voltage dependence of NMDA-R receptors?
- 40 to +40 linear
- 40 to -120 currents get smaller
removing Mg causes a linear response.
Mg show voltage dependent block of NMDA channel, blocks channel at more negative potentials.
?? rectification
128
L-Glu
| metabotropic glutamate types?
homodimers.
3 groups
groups 2 and 3 similar.
129
describe mechanism of group I mGlu-Rs
Group 1:
link to Gq, a subunit, increase PKC levels, phosphorylation occurs, closes K channels.
Tandem 2 pore domain K+ channels (K2P) close when phosphorylated → depolarization
NMDA-R current enhanced → ↑ responsiveness
Enhanced excitability
130
describe the mechanism of group II % III mGlu-Rs
link to Gi, inhibits adenylate cyclase decreasing PKA levels, leads to dephosphorylation or Ca channels. less functional channels.
P/Q- and N-type (CaV2.1-2) CaV channels (presynaptic) → ↓ release
L-type (CaV1.1-4) CaV channels (postsynaptic) → ↓ responsiveness
Reduced excitability
131
structure of GABA?
2a, 2b, y subunits.
GABAB
y, a, B, a, B
selective to Cl-
Eion of -70
hyperpolarisation if RMP >-70mv
132
GABA antagonists?
bicuculline (competitive, agonist binding site - red)
picrotoxin (Cl- channel - yellow)
133
glycine antagonist?
Strychnine
134
GABA receptor types?
GABA B1 and B2
need to form a dimer to be functional
B1 binds to GABA
B2 signals to G protein
135
antagonist for GABA?
Principal antagonists: CGP35348 (competitive agonist binding site)
136
agonist for GABA?
Principal agonist: baclofen (selective agonist)
137
GABA as treatment of narcolepsy?
Possible target for GHB (y-hydroxybutyrate) – treatment of narcolepsy, use by athletes (↑ growth hormone ) and drug of abuse 1990’s (“liquid ecstasy”)
138
targets for GABAB receptors?
target inward rectifier channels.
interacts with GPCR which increases GIRK channel function (K channel) through B subunit.
Gi
Postsynaptic → hyperpolarization.
Reduced excitability
Inhibit Ca channels.
direct/indirect pathways.
Gi inhibits adenylate cyclase, reduces cAMP, reduces PKA and leads to dephosphorylation of Ca channel.
P/Q- and N-type (CaV2.1 & 2.2) CaV channels (presynaptic) → ↓ release.
Reduced excitability
