Calcium Channels And Neurotransmitter
Release
There are many stages of neurotransmitter release that are calcium-
dependent, such as phosphorylation of synapsin in order to recruit
vesicles (kinase is calcium-dependent), and docking of vesicles. However,
the key step which is dependent upon calcium is exocytosis of the
neurotransmitter, which is triggered by the influx of calcium ions into the
pre-synaptic neuron.
Neuronal calcium homeostasis
A variety of calcium channels
and pumps act in concert to
maintain the extracellular
calcium concentration at 1mM,
keeping this greater than the
intracellular calcium
concentration of 100nM.
Voltage-gated Ca2+ channels
allow calcium ions into the
neuron in response to a
stimulus.
Ca2+ pumps are ATP
dependent pumps which pump
calcium ions out of the
cytosol/nerve terminal (to the
post-synaptic side) and hydrogen ions into the neuron.
Ca2+ exchanger exchanges calcium for sodium, utilising the strong
sodium gradient across the membrane to allow Na+ into the neuron and a
Ca2+ efflux.
Ca2+ permeable cation channels have permeability to calcium and
sodium ions, which are both allowed across the membrane into the
neuron.
Calcium buffering proteins exist such as calmodulin, and the principle
calcium buffering organelles in the synapse are the ER and mitochondria –
these buffer/prevent changes in calcium ion concentrations within cells.
This role of mitochondria is not typical in other cell types. Such buffering
organelles are trafficked along the cytoskeleton to the nerve terminal on
demand, and once here are important in buffering large changes in
calcium levels. They are important in disease, e.g. in many mitochondrial
diseases, defective mitochondrial activity means that its calcium buffering
capacity is lost, and the synapse becomes vulnerable.
Calcium imaging enables quantification of intracellular
calcium levels
Neurons are loaded with Fura-2 (AM) – a calcium sensitive dye which binds
to calcium (a calcium chelator) and fluoresces when exposed to UV light
, (neurons are therefore stimulated under a microscope to measure
changes in fluorescence).
Fura-2 is used for radiometric imaging – it has different excitation
emission properties depending on whether it is bound to calcium or not.
Neurons are excited at 340nm and 380nm, and the ratio of Fura-2
emission correlates with calcium level. Therefore this ratio is used to
quantify calcium changes in the synapse.
Images can be artificially coloured: blue
(low baseline calcium), green (modest rise
in calcium), red (high calcium levels).
Intracellular calcium levels change upon
stimulation – K+ leads to depolarisation and
opening of ion channels, giving the characteristic change in cellular
properties (?)
Genetically encoded calcium indicators CAMELEONS
There are more sophisticated ways of doing this, as developed by Roger
Tsien. He developed a genetic approach using GFP to measure calcium
changes in vivo, in living mammals.
GFP fused to calmodulin and M13 peptide of MLCK. Upon calcium binding
to calmodulin, a conformational change is induced which alters emission
properties of GFP.
Increasing the fidelity of neuronal Ca2+ exocytosis
coupling – localised calcium
entry
Free calcium is approx. 1Mm, and
resting intracellular calcium approx.
100nM. During membrane fusion, there
is tight regulation of these calcium
levels. The neuronal response to
calcium entry differs depending on the
proximity of the calcium-sensing
apparatus (SNARE complex) to the
source of calcium ions (voltage-gated
Ca2+ ion channel).
At 200nm distance:
1) Ca2+ concentration at SNARE
complex (calcium sensing machinery) = 5-10µM.
2) Rises and falls in 10msec (takes time for calcium ions to diffuse to
the sensing point).
3) Is at equilibrium with mobile buffers – buggers can cope very easily
with this sort of calcium change in the synapse
4) Strongly dependent on buggers; EGTA (chelating agent) as effective
as BAPTA (chelator, more selective for Ca2+)
5) Ca2+ determined by mean activity of several neighbouring channels
– to achieve an effective calcium concentration which is
meaningfully sensed, several channels operating together are
Release
There are many stages of neurotransmitter release that are calcium-
dependent, such as phosphorylation of synapsin in order to recruit
vesicles (kinase is calcium-dependent), and docking of vesicles. However,
the key step which is dependent upon calcium is exocytosis of the
neurotransmitter, which is triggered by the influx of calcium ions into the
pre-synaptic neuron.
Neuronal calcium homeostasis
A variety of calcium channels
and pumps act in concert to
maintain the extracellular
calcium concentration at 1mM,
keeping this greater than the
intracellular calcium
concentration of 100nM.
Voltage-gated Ca2+ channels
allow calcium ions into the
neuron in response to a
stimulus.
Ca2+ pumps are ATP
dependent pumps which pump
calcium ions out of the
cytosol/nerve terminal (to the
post-synaptic side) and hydrogen ions into the neuron.
Ca2+ exchanger exchanges calcium for sodium, utilising the strong
sodium gradient across the membrane to allow Na+ into the neuron and a
Ca2+ efflux.
Ca2+ permeable cation channels have permeability to calcium and
sodium ions, which are both allowed across the membrane into the
neuron.
Calcium buffering proteins exist such as calmodulin, and the principle
calcium buffering organelles in the synapse are the ER and mitochondria –
these buffer/prevent changes in calcium ion concentrations within cells.
This role of mitochondria is not typical in other cell types. Such buffering
organelles are trafficked along the cytoskeleton to the nerve terminal on
demand, and once here are important in buffering large changes in
calcium levels. They are important in disease, e.g. in many mitochondrial
diseases, defective mitochondrial activity means that its calcium buffering
capacity is lost, and the synapse becomes vulnerable.
Calcium imaging enables quantification of intracellular
calcium levels
Neurons are loaded with Fura-2 (AM) – a calcium sensitive dye which binds
to calcium (a calcium chelator) and fluoresces when exposed to UV light
, (neurons are therefore stimulated under a microscope to measure
changes in fluorescence).
Fura-2 is used for radiometric imaging – it has different excitation
emission properties depending on whether it is bound to calcium or not.
Neurons are excited at 340nm and 380nm, and the ratio of Fura-2
emission correlates with calcium level. Therefore this ratio is used to
quantify calcium changes in the synapse.
Images can be artificially coloured: blue
(low baseline calcium), green (modest rise
in calcium), red (high calcium levels).
Intracellular calcium levels change upon
stimulation – K+ leads to depolarisation and
opening of ion channels, giving the characteristic change in cellular
properties (?)
Genetically encoded calcium indicators CAMELEONS
There are more sophisticated ways of doing this, as developed by Roger
Tsien. He developed a genetic approach using GFP to measure calcium
changes in vivo, in living mammals.
GFP fused to calmodulin and M13 peptide of MLCK. Upon calcium binding
to calmodulin, a conformational change is induced which alters emission
properties of GFP.
Increasing the fidelity of neuronal Ca2+ exocytosis
coupling – localised calcium
entry
Free calcium is approx. 1Mm, and
resting intracellular calcium approx.
100nM. During membrane fusion, there
is tight regulation of these calcium
levels. The neuronal response to
calcium entry differs depending on the
proximity of the calcium-sensing
apparatus (SNARE complex) to the
source of calcium ions (voltage-gated
Ca2+ ion channel).
At 200nm distance:
1) Ca2+ concentration at SNARE
complex (calcium sensing machinery) = 5-10µM.
2) Rises and falls in 10msec (takes time for calcium ions to diffuse to
the sensing point).
3) Is at equilibrium with mobile buffers – buggers can cope very easily
with this sort of calcium change in the synapse
4) Strongly dependent on buggers; EGTA (chelating agent) as effective
as BAPTA (chelator, more selective for Ca2+)
5) Ca2+ determined by mean activity of several neighbouring channels
– to achieve an effective calcium concentration which is
meaningfully sensed, several channels operating together are
