1. nmj preparation* Note multiple release sites *Frog most common for earlier studies, this image is actually from a mouse

2. Katz & Miledi 1965 Frog neuromuscular junction No Calcium Focal application of calcium Focal application of a little less calcium Back to no Calcium postsynaptic response— release of neurotransmitter presynaptic action potential averaged responses Calcium is required for exocytosis

3. The “calcium hypothesis”: Ca 2+ entry into the axon terminal rapidly triggers exocytosis by binding to a “calcium sensor” for release Katz & Miledi 1967 These experiments proved it

4. 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 At ~20 , simplifies to: E m = membrane potential R = Gas Constant T = Absolute Temperature z = valance of ion (charge) F = Faraday’s Constant *AKA Reversal Potential, Equilibrium Potential

5. V = I x R I = g x (V m – V eq ) 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 1/R = g E Ca is very positive (>+100 mV), and g Ca 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 E Ca, reducing the driving force. For the calcium current: I Ca = g Ca x (V m – E Ca )

6. driving force (decreasing) calcium current (U-shaped) g (increasing) (membrane potential) resting potential

7. Katz & Miledi 1967 “suppression” potential is approached note the growing “off response” what is this? full suppression is achieved during pulse

8. Voltage-clamp recording of squid giant synapse Llinas, 1982

9. Release depends on [Ca] Ext 0.2 mM 0.25 mM 0.3 mMDodge & Rahamimoff, 1967 more calcium = more release

10. “Cooperativity” of the calcium dependence of release Dodge & Rahamimoff, 1967 multiple calcium ion must bind to trigger release R ≈ [Ca]/K Ca 1 + [Ca]/K Ca + [Mg]/K Mg n n is typically estimated between 2 - 4 NOT linear

11. How much calcium inside the cell is required for release 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 EPSCs -release of neurotransmitter is monitored as the postsynaptic response Schneggenburger & Neher, 2000

12. Only a little bit of intracellular calcium (9-10  M) is required to trigger release Schneggenburger & Neher, 2000 This is how much transmitter is released by an action potential

13. CC CC CC CC CC How fast is release? Very Fast less than 60  sec delay between start of I Ca and I post at 38  C Sabatini & Regehr, 1996 I Ca I post  time room temp fluorescent dyes current recording

14. 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?

15. Voltage-dependent calcium channels differ in their activation/inactivation ranges and kinetics (biophysical properties) Miller, 1987 note different time courses T-Type L-Type

16. Calcium channels differ in their sensitivity to pharmacological reagents L- dihydropyridines (nifedipine, nimodipine, enhanced by BayK); rarely controls release N- 1  M  Conotoxin fraction GVIA (  CTx-GVIA) T- 100  M Nickel (Ni 2+ ); mostly localized to dendrites Q- 1  M  Agatoxin fraction IVA (  Aga-IVA) 1.5  M  Conotoxin fraction MVIIC (  CTx-MVIIC) R- 5  M  CTx-MVIIC P- 30 nM  Aga-IVA All are blocked by 10 mM cobalt (Co 2+ ) and cadmium (Cd 2+ ) works pretty well, too.

17. We can use these tools to identify the channels that control neurotransmitter release Wheeler et al, 1994 (N- ty pe ) (Q- & R-) (P-)(N-)(N-) time (min ) Relative response size 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

18. How many calcium channels does it take to release a vesicle? Mintz et al., 1995 release no release release Ca channel vesicle [Ca] “profile”

19. Mintz et al., 1995 more than 100% 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  Aga-IVA used here blocks P- and Q-type channels)

20. 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 (~10  M) trigger release very quickly (<100  sec) 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

21. How does a vesicle fuse?

22. synaptic vesicle proteins Sudhof, 1995

23. SNARES and Synaptotagmin Littleton et al., 2001 -synaptosomal-associated protein of 25 kDa (SNAP-25) -syntaxin -vesicle-associated membrane protein (VAMP aka synaptobrevin) *NEM-sensitive factor (NSF) Soluble NSF* Attachment Receptors { (aka VAMP)

24. SNAREs were originally identified in yeast trafficking assays T-SNARES: V-SNARES:Golgi/PMvacuoleER/golgi plasma membrane vacuole Golgi/ER 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

25. Model of SNARE mediated fusion

26. Tetanus toxin and various serotypes of Botulinum toxin cleave SNARE proteins: 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 nb—tight form is insensitive to toxin

27. Effects of toxins on exocytosis (spontaneous EPSCs) (syntaxin)(SNAP-25)(vamp) Capogna et al., 1997

28. Broadie et al., 1995 TNT = Flies expressing TeTX throughout their nervous system to cleave VAMP (as with mice, some spontaneous release still present) Sys = Fly syntaxin knock-out Knocking out syntaxin abolishes all exocytosis

29. Geppert et al., 1994 Knocking out Synaptotagmin 1 (SytI) abolishes the fast Ca 2+ - triggered component of exocytosis, but not the slower Ca 2+ - dependent “asynchronous” component of release asynchronous release remains in SytI KO asynchronous release

30. 10 mM Ca 2+ 10 mM Sr 2+ Synchronous vs. asynchronous release asynchronous release synchronous release

31. Wild-type SytI KO Spontaneous mEPSCs are also unchanged in the SytI knock-out 1.9 minis/synapse/min 1.4 minis/synapse/min Geppert et al., 1994

32. 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?

33. Each C 2 domain binds 3 Ca 2+ ions w/affinities of 60  M, 400  M and >1 mM C 2 A domain binds phospholipids in a Ca 2+ -dependent & cooperative manner C 2 A domain binds syntaxin in a Ca 2+ -dependent manner (EC 50 =250  M) C 2 B domain mediates self-association of synaptotagmin I into multimers Synaptotagmin I: the calcium sensor?

34. Synaptotagmin I binds phospholipids (membranes) in the presence of calcium Earles et al., 2001 GST Control Synaptotagmin EGTA Ca 2+ Syt radiolabeled vesicle 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)

35. Ca 2+ binding to synaptotagmin I C 2 A domain: an “electrostatic switch”

36. How could Synaptotagmin I control Ca-dependent exocytosis? = SNARE complex Model I: Shao et al., 1997 Model II: Littleton et al., 2001 Either, neither, or both of these models could be correct (Could this be a fusion pore? )

37. 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 2 nd 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