Test 1 Flashcards
1
Q
Draw a neuron as an electrical circuit
A
look at picture
2
Q
Pumps/Transporters
ions are
Ionic movement =
A
separate charge
charged particles
electrical flow
3
Q
Active Transport by
Energy for this can be
A
ion pumps or transporters moves ions across the membrane
supplied by ATP, or the push from other ions
4
Q
Cells segregate
requires
A
ions: some are brought in, some are kept out
containing ions (membrane), releasing ions (ion channels)
5
Q
Cell membranes are
Hydrophilic
Hydrophobic
Lipids align in a
A
impermeable to ions
head: interacts with water, ions
tails: repels water, ions
bilayer: heads facing
out, tail to tail
6
Q
Being impermeable to ions, membranes
symbol, units
A
resist the flow of current
Resistance R; measured in ohms
7
Q
Conductance (g) is the
measured in
A resistor is a
A
inverse of resistance:
S (siemens)
conductor
8
Q
Ion Channels are proteins that
Some ion channels can
Flow of ions through ion channels means
Some have
Opening and closing creates
Open and close for
A
form a water-filled channel (pore) through the membrane
open and close
that current passes across the membrane
selective permeability for specific ions
ion-specific conductance
different reasons: voltage, ligands, etc
9
Q
Cell Physiology: Parallel circuits
Membrane has ion-specific
Variable conductance,
A
current has multiple paths through which to flow
conductances (g = 1/R) Creates a circuit of parallel conductances
such as ion channel opening and closing, is written as - arrow through the resistor
10
Q
Charge Neutrality
but
A
Intracellular and extracellular fluids are overall neutrally charged
(number of positive and negative charges is equal) total charge is neutral
Total summed concentrations of ions might be different across spaces! (intracellular is saltier)
11
Q
Concentration Gradient 2
A
Separation of molecules leads to a
concentration gradient, aka chemical gradient across the membrane
Concentration or chemical gradient
creates a force that pushes molecules to diffuse “down” the gradient, through ion channels
12
Q
Cell Physiology: Voltage
symbol and units
______ is an EMF
A
Another force that pushes ions, is the
E = Electromotive Force (EMF)
which pushes charges toward a balanced/neutral state
EMF, source denoted as E, has units of volts V
Membrane potential
13
Q
Each ion will be pushed by Membrane potential in a direction based on its
EMF source is a
symbol?
Because each ion is segregated
in different concentrations, each
has its
Because ion channels are ionspecific,
each ion has its own
Because this battery provides the
charges that flow through this
conductor, they are
A
charge
battery
parallel lines of different sizes
own EMF (battery)
resistor/conductor
in series
14
Q
Now that we have an EMF (a voltage, a pushing force), and a resistor/conductor (a path),
_____ is the movement
of charge
A
current will start to flow
Current
15
Q
Current =
which is amount of
measured in
which is
A
i
charge/unit time i = dq/dt
Amps = A
A = 1 C/s
(Coulombs per second)
16
Q
The amount of current that
flows depends on both:
Ohm’s law:
A
the strength of the push (the voltage) and
the resistance
how much current will flow; i = V/R
17
Q
For every ion there is an _______ series
Because ________ they are in parallel
A
EMF and conductance in
current can flow through multiple pathways
18
Q
Cell Physiology: Capacitance 2
key:
A
Lipid bilayer is highly impermeable to water and ions = Charged particles won’t pass through… but they like to interact with the
hydrophilic heads
Electrical charges interact across the membrane
membrane is thin enough that the charges can exert an attractive force across it
19
Q
Membrane _____ charge
Charge can flow
(even though
A
captures/stores
in or out of this region, making it a part of the circuit
charge isn’t flowing through the membrane)
20
Q
Capacitors parts 2
A
Two conductors + separated by a thin
resistor
21
Q
Capacitance (C) is:
A
C = Farads = (e A) / d
proportional to the area of the conductors (A)
inversely proportional to the distance separating them (d)
altered by the properties of the substance in
between e
22
Q
Importantly: The charge (Q) held is
So every time voltage (membrane potential)
changes, current is going to
symbol
A
equal to the capacitance times the potential (V) Q = C V
flow on or off the capacitor
two parallel lines of the same size
23
Q
- Effects physiology of the membrane:
- Effects our measurements of membrane physiology:
- Tells us things about the cell itself:
A
whenever membrane potential
changes, some current flow gets used up “charging” the membrane capacitance
whenever we change the membrane potential experimentally, current flows to the
capacitor
e and d are constant, so changes in C can tell us about things like size of the cell, release of vesicles, etc
24
Q
Cell Physiology: Ion Transport
for our purposes
must be on circuit diagram; sum of all transporter activity =
A
Pumps/Transporters separate charge = move ions
effectively constant.
current sources = two interlocking circles
25
Because of net charge neutrality, in each direction the
Note: the intracellular space is saltier (higher molarity). So
concentration gradients for positive and negative ions are balanced
so, there’s a greater push for both + and - ions to exit the cell.
26
ion channels are ion-specific!
give the membrane
For V to have a value:
So, for there to be a membrane potential, there
selective permeability
r must not be infinity and i must not be 0 (need unbalanced number of + and - ions flowing)
must always be SOME ion-specific channels open, conducting SOME current
27
In neurons at rest there are
which is
but these are ____ so no ____
always channels open that allow K+ to pass through
constantly replenished by ion pumps/transporters
These pumps are also pumping other ions in and out, so overall produce no net current themselves
28
Positive ions are flowing out. Negative ions
Negative ions trying to follow
Thus, near the membrane is a
try to follow, but can’t (membrane & channel are impermeable to them)
stick by the inner side of the membrane, apposed to positive charges outside
thin gradient of ions: negative inside, and positive outside
29
What about charge neutrality? 2
The number of ions crossing the membrane is minuscule compared with the total number in intra and extra-cellular space
The area of this charged gradient is tiny, and the overall net charge of the solutions is neutral
30
Membrane Potential exists because: 3
1. selective membrane permeability for K+ (R ≠ ∞)
2. EMF/concentration gradients push K+ out of the cell, so current is flowing (i ≠ 0) across the membrane
3. constant charge neutral replacement of
intracellular K+
31
Why do ions go in different directions?
```
The balance between:
1. chemical gradient (concentrations)
and the
2. electrical gradient (EMF)
gives each ion an electrochemical gradient and an equilibrium potential
```
32
Balancing Gradients
Sometimes, they
Depending on concentrations and membrane potential the gradients may
The one with more
Both concentration gradient (aka chemical)
and electrical gradient (membrane potential)
push ions to move across the membrane
push the same direction
push against each other
push dictates direction of flow
33
This is the EQUILIBRIUM POTENTIAL
(note: ions still
Each ion has its own
EQUILIBRIUM POTENTIAL
At specific membrane potentials, the gradients push ions with equal force in opposite directions
continue to diffuse back and forth, they just do so with equal probability in both directions)
proportional to intra- and extracellular concentrations
34
Equilibrium potential label and units
(labeled E, meaning it is an EMF and will have units of volts)
35
When membrane potential Vm = Eion
so also called ____ because ___
Equilibrium potential can be calculated with the
there will be no net flux of that ion across the membrane
Equilibrium potential is also called the REVERSAL POTENTIAL ; (because when if the membrane potential moves passed it, the direction of that ion’s flow reverses from in to out, or out to in)
Nernst Equation
36
Nernst Equation
Compare reversal and membrane
potentials, understand
_________:
membrane potential relative to the reversal potential
Eion = (58/z) * log([ion]out/[ion]in)
which direction ion will move
Direction of ion movement depends on
37
The difference between the membrane potential and the reversal potential 2
As the membrane potential (V) gets
farther away from the reversal potential
(E), the
is the DRIVING FORCE (U)
U = Vm - Eion
driving force gets bigger
38
The SIGN of the DRIVING FORCE (+ or -) tells you which
The AMPLITUDE of the DRIVING FORCE tells you how
modification of Ohm’s law:
direction the ion will flow
big the current for that single ion (Iion) will be
Iion = Uion/R
39
RESISTANCE 2
Thus, the membrane is
CURRENT
1. Ion channels have ion-specific permeability
and
2. At any time, some ion channels are open some are not
differentially permeable to different ions
1 Ions are differentially distributed so each has its own equilibrium potential and driving force
2 Each ion is passing current as it travels through ion channels,
and will proportionally contribute to voltage
40
Membrane potential is related to a combination of each ion’s 2
The contribution of a given ion is weighted by the relative
use
1. PERMEABILITY
and
2. REVERSAL POTENTIAL
permeability (the ratio) of the membrane to all the ions (p)
GHK equation
41
GHK equation
Vm = 58 * log(pk[K]o + pna[Na]o + pcl[Cl]i / pk[K]i + pna[Na]i + pcl[Cl]o)
42
Ions with higher permeability will have
i.e., membrane potential will be pulled toward the
greater influence on membrane potential
reversal potential of the dominant ion
43
GHK is a good model, but not )
Importantly, relative permeability is actually
perfect for biology (can’t take into account technical things like ionic interactions,
time-dependent and rectifying channels, etc
very hard to measure
44
parallel conductance model: replaces (2)
equation
replaces permeability with conductance
(easier to measure)
replaces Nernst ratio with reversal potential
Vm = (gkEk+gnaEna+gclEcl) / (gk+gna+gcl)
45
Driving force, Uion is the
The SIGN of the DRIVING FORCE (+ or -) tells you which
ion-specific voltage
direction the ion current will flow
46
+ Driving force
- Driving force
- Driving force (for - ions)
current flows outward
positive ions leave the cell
current flows inward
positive ions enter the cell
current flows inward
negative ions leave the cell
47
p are ____numbers that are just _____
unit-less; relative to each other
48
What’s an ion channel?
Protein that forms an aqueous pore through the membrane
49
Hetero-oligomers
Homo-oligomers
Single Polypeptide
Auxiliary Subunits
Made up of multiple individual protein subunits encoded by different genes
Made up of multiple, identical protein subunits, encoded by one gene
One gene encodes one big protein
don’t form the pore, but interact/ modulate/ traffic
50
Subunit
Motifs
Multiple transmembrane
(individual gene/protein): on its own, doesn’t
form a pore
sections of a subunit
motifs within each subunit
51
Ion Channels - Selectivity Filter 2
Filter cations and anions based on location of charged amino acids within the pore
Filter different ions of the same charge based on size and how they interact with water and amino acids while passing through the pore
52
Ion Channel Activation States
transitions?
closed, open, inactivated
(bidirectional transitions between these) - probabilistic
53
What is all that nontransmembrane stuff?
Inactivation domains, subunits interact, bind ligands, phosphorylation sites, bind other proteins, etc
54
State Changes During Stimuli 2
Even ideal stimuli often cause only transient opening of ion channels
Sometimes they will go through state transitions more than once
55
Why do currents always look square? 3
1. Most channels are either open or closed, current is all or none,
on or off
2. State transitions are extremely fast (no ramping up to full current)
3. When open, ion channels have a set single channel conductance
56
When closed or inactivated,
When open,
gchannel = 0, so Ichannel = 0
gchannel is set, so Ichannel is dictated by Uion
57
Channel opening is probabilistic =
Changing conditions (e.g., giving a stimulus) changes
At any given time, the channel can switch between states (open, close, inactivated)
the probabilities of certain state switches
58
Behavior of a single channel isn’t predictable, but…
These models make a very good prediction of how
Record enough channels, you can make a
model of these probabilities
populations of channels behave
59
In a cell, the total current is the
The summed current gets its shape
from the
sum of many, many single channels
sum of many probabilistic
openings and closings
60
To measure electrical changes across the membrane (i.e., voltage or current flow), we need to get access to
(The ground electrode is
both sides of the membrane
just a wire
stuck in the salty extracellular
solution, essentially a direct
connection to the circuit)
61
How do you get an electrode inside the cell?
Old school method #1:
Old school method #2:
use cells that are so big you can put a
wire right into them; Squid Giant Axons
Sharp Electrodes that can spear into a cell
62
Sharp Electrodes
Good:
Bad:
allows you to get an
electrode inside most normal-sized
neurons
pokes a hole in that neuron,
ions can leak out between the
electrode and the membrane
63
What is an Electrode?
Option 2: Glass capillary tubes (5)
```
To record membrane responses,
the electrode must be completely
insulated, there can be no other
path for current to flow, except
through the membrane
```
~When melted and stretched, a glass tube will maintain the same ratio of outer diameter:inner
diameter
~Stretched to a tiny diameter and then broken
~Filled through the top with solution that mimics intracellular fluid
~Wire threaded through the top so that it dips into the solution
~The glass “electrode” is then poked through the membrane
64
Patch Clamping 2
Forms a 3
~Blunt glass electrode is touched to the
cell membrane
~Membrane lipids stick tightly to the
glass
```
“Gigaohm seal”
~no ions can flow between the glass
and the membrane
~only route for current flow is
through channels in the membrane
```
65
Cell-attached recording:
whole cell recording
Excised patches (“pulling patches”):
attach to outside; fine for
some things, but current has to
flow through the membrane twice
Break patch of membrane
complete control of Vm, but
everything inside the cell is washed away
66
Outside-out patch
Inside-out patch
Difference?
Extracellular side exposed
Intracellular side exposed
e.g., you can test how a drug or protein binds and
modulates channel via intra- or extracellular domains
67
With amplifier, experimenter can control
(i.e., CLAMP) either
The third variable of Ohm’s
law, which we can’t directly
control, is RESISTANCE
voltage or current
This is what the
membrane/ion channel
does and what we measure
indirectly
68
VOLTAGE CLAMP
CURRENT CLAMP
Control Voltage
membrane Resistance changes
record Current
Control Current
membrane Resistance changes
record Voltage
69
Voltage Clamp Purpose:
How will you know when voltage clamp
has been used in an experiment?
~Inward current: 3
Outward current: 3
voltage clamp is great to isolate single channels or types of channels, and study how they behave
The data recorded will be currents
(labeled in amps, usually pA or nA),
and will often be graphed as a
function of time or voltage
positive ions moving in, negative ions moving out
-V-clamp recordings show inward currents going downward
~If Vm isn’t clamped, inward currents will make Vm more positive
(“depolarize” the cell)
positive ions moving out, negative ions moving in
-V-clamp recordings show outward currents going upward
~If Vm isn’t clamped, outward currents will make Vm more negative
(“hyperpolarize”
70
Amplifier controls the
FORCING FUNCTION:
Typical voltage clamp experiment:
voltage or current
between the two electrodes
how you change the aspect that you’re
clamping (voltage or current)
voltage steps
71
Uion = Vm - Eion 2
Slope of this line is
This is
changes linearly; gion is constant
SINGLE CHANNEL CONDUCTANCE
voltage independent ion channel behavior
72
Voltage Dependent Channel Behavior
Open probability
73
Excised patches are tiny Good: ____ Bad: ______
Whole cell recording: Good: ______
Bad: _______
single channels; how the cell actually
behaves
let’s you study the entire mix of a cell’s ion channels, how they respond and interact;
----Volume of the glass recording pipette is
huge compared to volume of a cell, so the
real intracellular solution is quickly replaced
with the artificial solution within the pipette
74
Bigger membrane patches, cells with high expression, and whole-cell
recordings are likely to record
many ion channels rather than single channels = Macro Currents
75
Single Channel recordings 2
Macro Current recordings 3
- always look square because the channel is either open and conducting current or closed
- opening and closing is probabilistic; makes it hard to tell by eye how channels will effect the cell
- lose their square-ness
- give you a better idea of what the channels will do for the whole cell
- get their shape from the probabilistic opening and closing of many single channels, summed together
76
Specific antagonists (or agonists) are used to
Add tetraethyl ammonium (TEA)
Add tetrodotoxin (TTX)
isolate the currents (channels) under study
Isolated Na+ currents
Isolated K+ currents
77
Quantifying Voltage Clamp
1. Amplitude increases (more channels activate with each step)
2. Amplitude saturates (all channels are activated)
3. Amplitude decreases (decreased driving force)
4. Amplitude reverses sign (passes reversal potential)
78
Quantifying Voltage Clamp graphs 2
I/V curve
Activation Curve (Ip/Ipmax vs Vm)
79
I/V curve linear section of function:
indicates
that conductance isn’t changing,
only driving force is changing)
80
Current Inactivation 2
go from some voltage to activating voltage (i.e.+50) - measure current
show change in current when change first voltage (closer to activating voltage = less current ---- less activation)
81
Why is direct permeability hard
| to measure? 2
```
Because intra- and extracellular
solutions contain many
different ions, we can only
measure current flow, but we
can’t tell which ions are the
ones flowing
```
Ratios of permeability
(IonA:IonB) are measured by
replacing the bath solution as
best we can
82
Current Clamp set up
Purpose:
How will you know when current
clamp has been used in an
experiment?
```
Current is held constant or changed
with forcing functions
Resistance changes (channels open)
Vm changes are recorded
```
In current clamp we see
how the cell behaves, how groups
of ion channels interact
The units of data recorded will be
volts (usually mV), plotted as a
function of current (amps), time,
etc.
83
Same shapes of forcing functions used in V-clamp can be
Responses to simple current clamp
protocols can
used in I-clamp
distinguish different cell types
84
Technical Considerations: Current flow across a membrane
The membrane is a capacitor: charge (Q) held is equal to the
every time voltage (membrane potential)
changes, current is going to
isn’t straightforward
capacitance times the potential (V) => Q = C V
flow on or off the capacitor
85
In a passive membrane (where ion channels aren’t voltage-gated) Ipassive =
it takes time for the capacitor (Vc) to charge to Vm so
Vm/Rm + Cm*dVm/dt
Ic has a big instantaneous
change that gradually fades
away as Vc approaches Vm
86
In a membrane with voltage-gated channels
usually have the passive and capacitor currents subtracted out = leave just the current through the voltage gated channel
87
Capacitance has
In V-clamp, we see it as an
In I-clamp, capacitance dictates
how
physiological implications
artifact and get rid of it
quickly the membrane can
respond
88
How quickly the membrane de- or hyperpolarizes, depends on
tau tells you if your
Lower R (more open channels) AND/OR Lower C (smaller cell)
```
Higher R (fewer open channels) AND/OR
Higher C (bigger cell)
```
Resistance and Capacitance
cell is fast or slow; tau = RC
faster
slower
89
Effectiveness of voltage CLAMP is
As current leaks out through
membrane channels, the voltage
of the membrane is
The (in)ability to control membrane
voltage at a distance from the
pipette is called
limited
less controlled
SPACE CLAMP
90
More membrane =
More channels =
```
Membrane extensions (processes)
like dendrites and axons are
```
```
Space clamp (voltage control) can
be improved by
```
more leak
more leak
harder to control
pharmacologically
blocking channels you aren’t
interested in studying
91
____3____ turn different subcellular
structures into “compartments”
Different compartments can have
Just like not all neurons are the
same, not all parts of an
Cell geometry, membrane
capacitance, and ion channels
combined
independent physiologies
individual neuron are the same
92
Electrical compartments from
Differential Ion Channel Expression
93
The Action Potential 3
Basic unit of neuronal electrical signaling
All or none signal
Digital - discrete units of information
94
Reaching Threshold 2
Threshold =
how reach?
Membrane potential begins to
depolarize
Voltage-gated Na+ channels
(VGSCs) begin to activate
(may cause an acceleration of
depolarization)
not so much a set
number as a tipping point
VGSCs activation,
channels open, the membrane
depolarizes, so more VGSCs open
(positive feedback)
95
The Depolarizing Phase
Membrane becomes highly
permeable to Na+, pulling Vm toward
ENa (+58 mV
96
The Repolarizing/Hyperpolarizing Phase 3
```
VGSCs INACTIVATE (time- and
voltage-dependent)
```
Voltage-gated K+ channels open
Membrane becomes highly permeable to K+, not to Na+, pulling Vm toward EK (-97 mV), overshoots Vrest
97
Return to Rest 2
Voltage-gated channels
DEACTIVATE (time- and voltage dependent)
Membrane becomes moderately
permeable to K+, returns to Vrest
98
draw Time course of ionic conductances
look at thing
99
Refractory Period 3
Recovery from VGSC inactivation is time-dependent
Spikes won’t fire while VGSCs are inactivated (refractory period)
Threshold remains shifted for longer period
100
Ion Channel Families
_____ voltage-gated Na+ channels, ______ of voltage-gated K+ channels)
Many types of ion channels, grouped into families
10 types of; several separate families
101
Ion channel family members can have differences in: 4
Gives neurons an
1Gating - voltage sensitivity, state switching probabilities, kinetics, permeability, conductance
2Which cell-types they are expressed in 3Subcellular distribution
4Protein/protein interactions, binding partners, modulation/phosphorylation
enormous range of flexibility in how they can
respond to stimuli
102
Spike shape/pattern is as
Differences largely due to
diverse as cell types
differential channel expression and subcellular distribution
103
Axons are 2
AIS, aka:
highly organized compartments; ion channels anchored
Axonal hillock, initial segment, spike trigger zone, spike initiation zone
104
The Axon - Initial Segment (AIS) 3
High Na+ channel density is important
By concentrating Na channels within a small region, an AP can be evoked without having to coat the entire neuron with channels
Gives better control of AP generation, rather than spikes randomly firing off due to depolarization of individual dendrites
105
The Axon - Myelin 2 types
AP propagation is
Myelin increases ____ and decreases ___
More current can
Saltatory conduction -
Current flow easily
Schwann cells in the PNS; Oligodendrocytes in the CNS
fast and efficient with myelin
resistance and decreases capacitance
directly flow down the axon, to depolarize the next Node of Na+ channels
action potential “jumps” from node to node
depolarizes the next node to threshold and regenerates the action potential
106
The Axon - Nodes of Ranvier 3
Na+ channels cluster in node
K+ channels cluster in paranode
Nodes are highly structured compartments
107
The Axon - Demyelination 2
Demyelinating Diseases
Without
Loss of myelin decreases or blocks AP propagation
Myelin may not develop properly, or might form and then be removed
Demyelination in the CNS - Removal of Myelin = Multiple Sclerosis - immune disruption of myelin
action potential propagation, there is no synaptic transmission
108
The Presynaptic Terminal: Active zone
The “synapse” might be
= area of contact where all the machinery for vesicle release coexists
a larger area where neurons are adjacent,
with a smaller area (or areas) making up the active zone
109
Convergence =
Divergence =
number of presynaptic neurons that project to a single postsynaptic neuron
number of postsynaptic neurons that a single presynaptic neuron projects to
110
Purkinje cells of the cerebellum:
Parallel fibers: (axons from granule cells) 3
Climbing fibers: (axons from inferior olive) 3
```
-one axon makes 1-5 synapses with a
Purkinje cell (weak effect)
-200,000 axons contact one Purkinje cell
(extremely high CONVERGENCE)
-one axon may connect to 1000s of
Purkinje cells (high DIVERGENCE)
```
```
-1 axon makes 500 synapses with a single
Purkinje cell (powerful effect)
-one axon connects to one Purkinje cell
(minimal convergence)
-one axon may to connect to 1-5 Purkinje
cells (low divergence)
```
111
Electrical to chemical:
Action potential becomes intracellular Ca2+
112
Voltage Gated Calcium Channels 3
Presynaptic terminal channels are
largely N- and P/Q-type
single polypeptide
made of motifs
113
Presynaptic calcium channels tend to open only during
large deploarizations (AP not subthreshold)
114
To use Ca2+ as an intracellular signal, cells are
Neurons have strong Ca2+
Calcium Domains: Nanodomains
Synergistic activation
Microdomains
very effective at keeping intracellular Ca2+ low (otherwise, pathways Ca2+ activate
would be active all the time)
buffering: once Ca2+ comes in it doesn’t get very far before it is soaked up
Single channels, Local [Ca2+], no overlap
Channels closer mean higher local [Ca]
Multiple calcium channels
115
Why Calcium? 3
There isn’t nearly as much of it to begin with,
intracellular concentration can be further lowered
But, there’s enough for it to be biologically relevant
Divalent: interacts/binds in a fundamentally different way than most ions
116
Chemical to enzymatic:
intracellular Ca2+ activates protein
117
Calcium Sensors -
Many different____ proteins. The important thing, they all have: (2)
Synaptotagmins
The Syt family of proteins: presynaptic calcium binding proteins
Syt; one T = transmembrane domain and two C2 = Ca binding domains
118
Two Syt domains bind
______ bind and hold calcium ions
Syts are
multiple calcium ions
Negatively charged residues
transmembrane proteins in the synaptic vesicle membrane
119
Change in [Calcium] is
Everything must be in
To function, the vesicle/Syt complex
must be
local and fast
close proximity
right up against the membrane
to dip the Syt into the Ca domain
120
Enzymatic to mechanical:
activated protein has to move things around
121
Synaptobrevin
rab3:
(Syb, VAMP): vesicle side of the SNARE complex
localizes vesicle near calcium channels
122
The SNARE complex:
3 key components
holding the vesicle by the membrane
Synaptobrevins (Syb) are VAMPs (vesicle associated membrane protein, not all VAMPs are synaptobrevins), transmembrane proteins in the vesicle
SNAP-25 not transmembrane, but membrane anchored by palmitoyl side chains on cysteines, which are attached to cytosolic membrane
Syntaxin (Synt) transmembrane protein in the cytosolic membrane
123
The SNARE complex 2
Form quaternary twisted structure of 4 alpha-helices (SNAP-25 contributes two)
Physically link the vesicle right next to the membrane
124
Forming the SNARE complex 3
Munc18 (STXBP1, syntaxin binding protein) soluble protein
Binds Syntaxin-1, modulates interaction with SNAP-25 and Synaptobrevin, reduces inappropriate complex formation
Absolutely necessary for synaptic transmission
125
Vesicle release - Priming:
Vesicle approaches
This
Syt
Vesicle is now
Complexin
Munc-13
Vesicle is now
Vesicle approaches membrane, v-SNARE (vesicle) and t-SNARE (terminal)
twist into SNARE complex
This pulls vesicle into close proximity to the membrane
Syt in range of Ca2+ micro-domains
Vesicle is now part of “readily releasable pool” - competent for Ca2+-induced release
Complexin binds to, completes SNARE
complex
Munc-13 binds to RIM
Vesicle is now right up against membrane
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Calcium-induced
Calcium binding causes 2
conformation changes
- conformational change in Syt1
- Pulls and warps the membrane
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Opening the Pore
Syt1 binding with Ca changes its shape, causes alpha helices to “zipper”, twist together
Membranes touch, lipid layers connect
Water-filled pore opens
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Neurotransmitter Release
neurotransmitter concentration very high inside vesicle
open pore = large amount of NT diffuse out
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3 Key Synaptic Vesicle Proteins for Release/ “Docking” the Vesicle Near Calcium Channels
Rab3 localizes vesicles near Ca channels, soluble protein modified to bind vesicle membrane
Munc13,
RIM (rab3 interacting molecule),
RIM-BP (Rim Binding Protein): soluble proteins form the scaffold
Ca2+ channel both RIM and RIMBP bind directly to C-terminal domain
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What happens to the vesicle? (3)
Any of the 3 mechanisms can occur,
depending on
1. Kiss-and-run: pore opens, re-closes, vesicle structure/proteins retained, just needs
to be refilled
2. Vesicle collapse: loses shape, becomes part of plasma membrane, proteins diffuse away
3. Ultrafast Endocytosis: moves out of active zone, loses some shape, but proteins retained, vesicle reformed (via endosomal pathway)
which proteins are expressed, and altering what gets released from the vesicle
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NSF -
Clathrin
unfolds and releases SNARE complex for recycling
coats vesicle, allows vesicle membrane to be pulled away from plasma membrane and recycled
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Recycling Synaptic Vesicles
Readily retrievable pool pathway (Kiss-and-run)
Direct pathway (Vesicle collapse)
Endosomal pathways (Ultrafast Endocytosis)
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Ending the Cycle:
Multiple mechanisms
what are 4 of the mechanisms
Calcium Clearance
rapidly clear calcium out of the terminal
- Ca buffers
- PMCA pump (plasma membrane pump) atp
- SERCA pump (sarco/endoplasmic reticulum) atp
- Na-Ca transporter
