Calcium in the Axon Terminal

What is the sequence of events in chemical transmitter release?

Action potential→ nerve terminal depolarization→ activation of voltage-gated calcium channels→ calcium enters down a STRONG gradient→ Calcium triggers transmitter release→ exocytosis of neurotransmutter→ Transmitter crosses synaptic cleft→ transmitter binds to receptor→ postsynaptic conductance change→ action potential

What are the three important points regarding intracellular and extracellular Ca++ concentrations?

1. “free” intracellular calcium is kept at very low concetrations
2. thus, there is a large concentration gradient for calcium
3. this makes calcium an ideal signaling molecule

What is a dissociation constant? (Kd)

1. measure of strength of binding affinity
2. calculated as the ratio of the unbinding rate (Koff) duvuded by the binding rate (Kon)
3. Kd value indicates the concentration at which 50% of binding sites are occupied
4. Kd and affinity are inversely related

What is the Plasma Memmbrane Ca-Mg-ATPase (PMCA)

* transporters that move Ca++ out of the cytoplasm
* utilizes ATP to move Ca++ against its concentration gradient (primaru active transport)
* Mg++ is a co-factor
* internal side of transporter has high affinity for Ca++ (compared to NCX): requires 300-500nM Ca++ to be activated

Na-Ca exchanger (NCX)

* transporter that move one Ca++ ion out for every three Na+ ions in
* utilizes energy from moving Na+ down its concentration gradient to move Ca++ against its concentration gradient (secondary active transport)
* only active breifly after a high-frequency burst of action potentials
* internal side of transporter has low affinity for Ca++ (compared to PMCA): requires 700nM-1microM of Ca++ to be activated

What are two intracellular calcium buffers?

* intracellular organelles that buffer calcium
* intracellular calcium binding proteins

What are the intracellular organelles that buffer calcium?

* endoplasmis reticulum
* mitochondria

What does the intracellular calcium binding proteinsdo?

Quickly reduce the concentration of free Ca++ ions in the cytoplasm

What does the RyR do?

induced calcium release (allows the calcium move down its concentration gradient into the cytosol)

What does do IP3 do?

it is a signalling system for calcium where calcium ends up being release into cytosol

What does STIM1 do?

proteins that “refill ER” and activates the “CRAC channels” to allow calcium to move down its concetration gradient and then into teh SERCA channels to then be pumped into the ER.

* STIM1 is activated when Ca conctration is ER is low

VDAC calcium transporter in the mitochondira:

* non-selective cation channel that is bi-directional
* depended on the concentration gradient of calcium within the mitochondira

mNCX transporter in mitochondira

works like the transporter described earleir into teh mitochondira (3Na for 1Ca)

MCU mitochondira calcium transporter in mitochondira:

Uniporter that moves calcium into the innger lumen of the mitochondira (down concentration gradient so no ATP used)

ETGA alien buffer for study of calcium control of transmitter release:

* high affinity buffer
* slow (binds well but very slow to sop up the calcium)
* the slow binding kinetics still allows for the postsynaptic response

BAPTA alien biffer to study calcium control of trasmitter release:

* high affinity and fast to bind
* the BAPTA sops up the calcium quickly and can prevent neurotransmitter release

Calcium entry is restricted to…..

active zone of the frog nerve terminal

What is calmodulin?

* CaM: short for “calcium modulated protein”
* trype of calcium binding portein found in neurons and other cells

What is Green FLuorscent calmodulin proteins? (GCaMP)

will fluoresce when Calcium is bound to them

The pulse experiment identified that Calcium is necessary for….

* vesicle release
* observed with removing calcium from bath and pulse calcium onto terminal
* and add ionic blocker of calcium channels
* identified that calcium must be present DURING stimulus to resupt in a response (calcium after stimulus evokes no response)

There is experimental evidence that calcium is sufficient for….

vesicle release.

* note that in the absence of action potential, voltage gated ion channel and everything→ calcium alone is enough to trigger vesicular release
* liposome= little vesicle of stuff to directly deliver the stuff to membrane on nerve terminal
* ionophore=channel that is permanently open only for calcium
* calcium alone shows release vesicles→ calcium is followed by a “runaway epp” that is a very large and long EPP response.