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nmj preparation*. Note multiple release sites. *Frog most common for earlier studies, this image is actually from a mouse. Calcium is required for exocytosis. averaged responses. No Calcium. presynaptic action potential. Focal application of calcium. postsynaptic response—

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Nmj preparation

nmj preparation*

Note multiple release sites

*Frog most common for earlier studies, this image is actually from a mouse


Calcium is required for exocytosis

Calcium is required for exocytosis

averaged responses

No Calcium

presynaptic action potential

Focal application

of calcium

postsynaptic response—

release of neurotransmitter

Focal application

of a little less calcium

Back to no Calcium

Frog neuromuscular junction

Katz & Miledi 1965


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The “calcium hypothesis”: Ca2+entry into the axon terminal rapidly triggers exocytosisby binding to a “calcium sensor” for release

These experiments proved it

Katz & Miledi 1967


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At the Nernst Potential*, the balance of electrical and diffusion tendencies creates an electrochemical equilibrium between the opposing chemical (concentration) and electrical forces—NO NET MOVEMENT OF IONS

Em = membrane potential

R = Gas Constant

T = Absolute Temperature

z = valance of ion (charge)

F = Faraday’s Constant

At ~20, simplifies to:

*AKA Reversal Potential, Equilibrium Potential


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The amount of current carried by a particular ion across a membrane is a function

of how many channels that are permeable to that ion are open (the “conductance”,

or “g”), and the “driving force” for that ion, that is, how far the membrane potential

is from the equilibrium potential for that ion

V = I x R

1/R = g

I = g x (Vm – Veq)

For the calcium current: ICa = gCa x (Vm – ECa)

ECa is very positive (>+100 mV), and gCa is voltage-dependent, so increasing membrane potential (depolarization) will open more and more calcium channels, increasing the conductance for calcium, but will also bring the membrane potential closer to ECa, reducing the driving force.


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resting potential

(membrane

potential)

calciumcurrent (U-shaped)

g (increasing)

driving force (decreasing)


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“suppression” potential

is approached

what is this?

note the growing

“off response”

what is this?

full suppression is achieved

during pulse

Katz & Miledi 1967


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Voltage-clamp recording of squid giant synapse

Llinas, 1982


Release depends on ca ext

Release depends on [Ca]Ext

0.2 mM

0.25 mM

more calcium = more release

0.3 mM

Dodge & Rahamimoff, 1967


Cooperativity of the calcium dependence of release

[Ca]/KCa

n

R ≈

1 + [Ca]/KCa + [Mg]/KMg

“Cooperativity” of the calcium dependence of release

multiple calcium ion must bind to trigger release

NOT linear

n is typically estimated between 2 - 4

Dodge & Rahamimoff, 1967


How much calcium inside the cell is required for release from mammalian cns neurons

How much calciuminside the cell is required forrelease from mammalian CNS neurons?

-giant presynaptic terminal (Calyx of Held) is filled with “caged” calcium

-a flash of light “uncages” the calcium; the brighter the flash,

the more calcium is uncaged

-a fluorescent calcium-indicator dye reports the concentration

of calcium in the terminal

-release of neurotransmitter is monitored as the postsynaptic response

EPSCs

Schneggenburger & Neher, 2000


Only a little bit of intracellular calcium 9 10 m m is required to trigger release

Only a little bit of intracellular calcium (9-10 mM) is required to trigger release

This is how much transmitter

is released by an action potential

Schneggenburger & Neher, 2000


How fast is release

C

C

room temp

C

C

C

D time

How fast is release?

fluorescent dyes

Ipost

current recording

ICa

Sabatini & Regehr, 1996

Very Fast

less than 60 msec delay between start of ICa and Ipost at 38C


Calcium channels controlling release

Calcium channels controlling release

There are many different subtypes of voltage-gated calcium channels in neurons, including:

L(onglasting)-, T(ransient)-, N(either)-, P(urkinje)-,

Q(cool letter, in the right region of the alphabet)-, and

R(esistant)-type channels

…and this doesn’t even include ligand-gated calcium channels!

How can we start to distinguish between them and

identify their roles (if any) in controlling synaptic transmission?


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Voltage-dependent calcium channels differ in their activation/inactivation ranges and kinetics (biophysical properties)

T-Type

note different

time courses

L-Type

Miller, 1987


Calcium channels differ in their sensitivity to pharmacological reagents

Calcium channels differ in their sensitivity to pharmacological reagents

L-dihydropyridines (nifedipine, nimodipine, enhanced by BayK); rarely controls release

N-1mM wConotoxin fraction GVIA (wCTx-GVIA)

T-100 M Nickel (Ni2+); mostly localized to dendrites

Q-1 M wAgatoxin fraction IVA (wAga-IVA)

1.5 M wConotoxin fraction MVIIC (wCTx-MVIIC)

R-5 M wCTx-MVIIC

P-30 nM wAga-IVA

All are blocked by 10 mM cobalt (Co2+) and cadmium (Cd2+) works pretty well, too.


We can use these tools to identify the channels that control neurotransmitter release

(P-)

(N-)

(N-type)

Relative response size

(Q- & R-)

time (min)

We can use these tools to identify the channels that control neurotransmitter release

Release at these hippocampal CA3-CA1 synapses is controlled by N-, and Q and(/or)

R-type calcium channels; P-type channels do not control release at these synapses

Wheeler et al, 1994


How many calcium channels does it take to release a vesicle

How many calcium channels does it take to release a vesicle?

[Ca] “profile”

release

release

no release

Ca channel

vesicle

Mintz et al., 1995


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Non-additive effects of toxins on release suggests that multiple calcium channels must open to trigger vesicle fusion at mammalian CNS synapses

(note-higher conc. of wAga-IVA used here

blocks P- and Q-type channels)

more than

100%

Mintz et al., 1995


Messages of the day

Messages of the Day

  • Intracellular calcium triggers release of neurotransmitter

  • Calcium ions act cooperatively to trigger release (calcium-release relationship is not linear)

  • Very small increases in intracellular calcium (~10mM) trigger release very quickly (<100msec)

  • Different types of voltage-dependent calcium channels control release of neurotransmitter

  • Entry of calcium through more than one calcium channel may be required to trigger release


How does a vesicle fuse

How does a vesicle fuse?


Synaptic vesicle proteins

synaptic vesicle proteins

Sudhof, 1995


Snares and synaptotagmin

{

SNARES and Synaptotagmin

-synaptosomal-associated protein of 25 kDa (SNAP-25)

-syntaxin

-vesicle-associated membrane protein (VAMP aka synaptobrevin)

Soluble

NSF*

Attachment

Receptors

*NEM-sensitive

factor (NSF)

(aka VAMP)

Littleton et al., 2001


Snares were originally identified in yeast trafficking assays

V-SNARES:

Golgi/PM

vacuole

ER/golgi

T-SNARES:

plasma

membrane

vacuole

Golgi/ER

SNAREs were originally identified in yeast trafficking assays

Only some combinations lead to fusion—leading to the hypothesis that SNAREs impart specificity to fusion reactions—and may be the “minimal machinery” required for vesicle fusion

McNew et al., 2000


Model of snare mediated fusion

Model of SNARE mediated fusion


Tetanus toxin and various serotypes of botulinum toxin cleave snare proteins

Tetanus toxin and various serotypes of Botulinum toxin cleave SNARE proteins:

nb—tight form is

insensitive to toxin

Syntaxin is cleaved by BoNT/C

SNAP-25 is cleaved by BoNT/A and E

Vamp (synaptobrevin) is cleaved by TeNT, BoNT/B, D, F and G


Effects of toxins on exocytosis spontaneous epscs

Effects of toxins on exocytosis (spontaneous EPSCs)

(vamp)

(SNAP-25)

(syntaxin)

Capogna et al., 1997


Knocking out syntaxin abolishes all exocytosis

Knocking out syntaxin abolishes all exocytosis

TNT = Flies expressing TeTX throughout their nervous system to cleave VAMP (as with mice, some spontaneous release still present)

Sys =Fly syntaxin knock-out

Broadie et al., 1995


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Knocking out Synaptotagmin 1 (SytI) abolishes the fast Ca2+-triggered component of exocytosis, but not the slower Ca2+-dependent “asynchronous” component of release

asynchronous release remains in SytI KO

asynchronous release

Geppert et al., 1994


Synchronous vs asynchronous release

Synchronous vs. asynchronous release

synchronous release

asynchronous release

10 mM Ca2+

10 mM Sr2+


Spontaneous mepscs are also unchanged in the syti knock out

Spontaneous mEPSCs are also unchanged in the SytI knock-out

Wild-type

1.9 minis/synapse/min

SytI KO

1.4 minis/synapse/min

Geppert et al., 1994


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Synaptotagmin I is required for fast Ca-triggered synaptic vesicle exocytosis, but not slower Ca-dependent asynchronous release or Ca-independent spontaneous release—could synaptotagmin I be a fast calcium sensor?


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Synaptotagmin I: the calcium sensor?

  • Each C2 domain binds 3 Ca2+ ions w/affinities of 60 mM, 400 mM and >1 mM

  • C2A domain binds phospholipids in a Ca2+-dependent & cooperative manner

  • C2A domain binds syntaxin in a Ca2+-dependent manner (EC50=250 mM)

  • C2B domain mediates self-association of synaptotagmin I into multimers


Synaptotagmin i binds phospholipids membranes in the presence of calcium

EGTA

Ca2+

GST Control

Synaptotagmin

Synaptotagmin I binds phospholipids (membranes) in the presence of calcium

radiolabeled

vesicle

Syt

heavy beads coupled to Syt are

mixed with radioactive lipid vesicles,

then spun down in a centrifuge, bringing

along anything the Syt has bound to

(unbound lipid vesicles stay in solution)

Earles et al., 2001


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Ca2+ binding to synaptotagmin I C2A domain:

an “electrostatic switch”


How could synaptotagmin i control ca dependent exocytosis

= SNARE complex

How could Synaptotagmin I control Ca-dependent exocytosis?

Model II: Littleton et al., 2001

Model I: Shao et al., 1997

)

(Could this be a fusion pore?

Either, neither, or both of these

models could be correct


Messages of the day1

Messages of the Day

  • Presynaptic SNAREs are the minimal machinery required for both calcium-dependent and spontaneous fusion

  • Synaptotagmin is currently the best candidate for the fast Calcium Sensor triggering calcium-dependent synchronous release

  • A 2nd Calcium Sensor controls asynchronous release

  • The mechanisms underlying SNARE and synaptotagmin mediated fusion are hot topics of research—likely to be worked out within the next 5 years


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