Selective disruption of the mammalian

secretory apparatus enhances or eliminates

calcium current modulation

in nerve endings

Eugene M. Silinsky* PNAS 2008, 105-17: 6427

Martha Eugenia Ramirez-Dominguez - 092010 - MARK’S LAB JOURNAL CLUBUCSF - SFGH

Docking: specifically attaches vesicles to the active zone; Priming: makes the vesicles competent for Ca2+ -triggered release and

may involve a partial fusion reaction; Fusion: final Ca2+ -regulated step that completes fusion.

Synaptic vesicle exocytosis occurs in consecutive steps: M

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Microdomains of high [Ca]I occur at the pore exit, because Ca2+ enter the channel faster than they can diffuse into the surrounding cytoplasm.

Why do calcium channels (Cavs) interact with proteins thought to be located at the

interface between a docked synaptic vesicle and the plasma membrane?

Primed fusion-ready vesicles that are not linked to CaVs are much less likely to release their contents when an action potential invades the nerve terminal.

It follows on from this hypothesis that regulatory processes which modulate interactions between channels and synaptic core complexes could

play an important role in the plasticity of presynaptic events.

Molecular links between CaVs and the exocytotic machinery would guarantee that the calcium trigger initiating vesicle fusion is located within a restricted zone where the appropriate [Ca]i is attained upon channel activation.

Synaptic vesicle fusion requires relatively high 50-100 uM [Ca]i

only attained close to the inner mouth of the activated CaVs.

Synprint Site

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Synaptotagmin Synaptotagmin : : Ca2+ -binding protein, essential for Ca2+-triggered release and probably serves as the Ca2+-sensor in fusion.

Rab3: limits the number of vesicles that can be fused as a function of Ca2+

in order to allow a temporally limited, repeatable signal.

Once membrane depolarization and Ca influx occur, conformational changes in the SNARE complex, along with other less well defined processes,

facilitate the fusion of the two membranes and release neurotransmitter into the synapse. Mar

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Skeletal Neuromuscular Junction

G protein coupled Receptors (GPCR) :

modulated by Ca2= entrance through Cav- Channels&

Interaction with Secretoty Apparatus

Adenosine

GPCR

Inhibit Neurosecretion

Neuromuscular depression

Agonist

1) A1 – Adenosine Receptor Activation A1R(+) P/Q Cav Neurotransmission

Neuromuscular Depression

Adenosine effect on Secretory Complex by Cavs ?

Anfibia NOMouse YES

2) By BoNTx assays SNARES important role in mediating effects of Adenosine at

mouse motor nerve

So, there is an interconection between A1R – P/Q Ca channels

mediated by SNAREs ?

Ach release

The secretory apparatus and the cleavage sites for BoNtxs.

Cav channels N-type and P/Q-type contain a

Synaptic Protein Interaction (synprint) site within the II–III loop that assembles with synaptic proteins

such as Syntaxin (Sx), SNAP-25 (SN) and Synaptotagmin (Sg)

This association couples synaptic vesicles and the source of extracellular calcium and

serves as a regulatory element

by which

synaptic proteins may control calcium channel function .

Synprint

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Isolated phrenic nerve hemidiaphragm from Rab3A−/− mutant mice

Calcium currents were quantified by measuring voltage changes in the perineural space

The perineural recording electrodes were filled with normal physiological salt solution (3–10 MΩ) and positioned near small axon bundles at the heminode.

The electrophysiological correlates of Ach secretion (EPPs) were also recorded to monitor the elimination of Ach release.M

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The effects of Adenosine in the Rab3A-/- mutant with and w.o. BoNTx treatment

*9 aaNor

required

Presynaptic ionic currents

End-plate potentials Ach release

Sodium current

P/Q calcium current Cd++ & ω-agatoxin block

SxImportant target x Adenosin inhib

SN-25Coupling A1R to Cav inhib

***25 aa

17 aa required

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Increased efficacy of adenosine in the Rab3A-/- mutant after cleavage of the vesicle protein synaptobrevin with Botx/D.

Complete inhib of P/Q CavReversible

Prevent vesicle participate in priming with t-SNARES

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A model for the enhanced effects of adenosine before and after disruption of vesicle proteins.

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SV fusion is likely to be initiated by the change in conformation and/or electrostatic potential of a Ca2+ -sensing protein.

Similarly, limiting neurotransmitter release to the particular SV that has initiated the fusion process—out of a pool of fusion-competent vesicles—

requires a mechanism that excludes a complicated time-consuming cascade of protein- protein-interactions.

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• what are the clostridial neurotoxins doing that is so deadly? ‘Blocking neurotransmission’ is the answer which prompted many physicians

‘SNARE cleavage’ is the answer that stimulated many cell biologists to use clostridial neurotoxins as research tools for solving fundamental questions on secretion.

What will the next answer and application be?

How botulinum and tetanus neurotoxins block neurotransmitter releaseHow botulinum and tetanus neurotoxins block neurotransmitter release

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Jarvis & Zamponi (2005) Cell Calcium37, 5: 483-488Calcium in the function of the nervous system: New implications

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