Lecture 10: Calcium in the axon terminal

Sequence of events in chemical transmitter release

1. presynaptic action potential

2. nerve terminal depolarization

3. activation of voltage gated calcium channels

4. calcium enters down a strong gradient

5. calcium triggers neurotransmitter release

6. exocytosis of neurotransmitter

7. transmitter crosses synaptic cleft

8. transmitter binds to postsynaptic receptor

9. postsynaptic conductance change

10. post synaptic action potential

Intracellular and extracellular calcium concentrations

• not very high extracellular calcium concentration

• “free” intracellular calcium is kept at low concentrations

  • calcium that is free to do anything(not removed by buffers/organelles)

• thus, there is a large concentration gradient for calcium since intracellular calcium concentration is so low

• this makes calcium an ideal signaling molecule: influx of even a little is noticeable

• extracellular calcium is less than extracellular potassium so extracellular calcium isn’t too important → intracellular calcium must be low

Dissociation constant(KD)

• a measure of the strength of binding affinity

  • more affinity = better two things bind

• calculated as the ratio of the unbinding rate(Koff) divided by the binding rate(Kon)

• KD value indicates concentration at which 50% of binding sites are occupied

• KD inversely related to affinity

• KD>1: low binding

• KD<1: high binding

Maintenance of calcium concentration: transporters

plasma membrane Ca-Mg-ATPase(PMCA)

• transporter that moves calcium out of cytoplasm: keeps intracellular concentration low

• uses ATP to move calcium against its concentration gradient

  • ATP as energy source = ATPase = primary active transport

• Mg2+ is a cofactor: not transported but required

• internal side of transporter has high affinity for calcium compared to NCX so needs 300-500 nm calcium to be activated

  • high affinity=low KD=needs less concentration for 50% to be binding

calcium homeostasis

• active zone=location where vesicle is released

Maintenance of calcium concentration: transporters

Na-Ca exchanger(NCX):

• transporter that moves one calcium ion out for 3 sodium ions in

• utilizes energy from moving sodium down its concentration gradient to move calcium against its concentration gradient

  • energy from moving down its concentration gradient=secondary active transport

• only active briefly after a high-frequency burst of action potentials

• internal side of transporter has low affinity for calcium compared to PMCA: required 700nm-1micromolar calcium to be activated

Maintenance of calcium: intracellular calcium buffers

• intracellular organelles that buffer calcium:

  • endoplasmic reticulum

  • mitochondria

• intracellular calcium binding proteins

  • quickly reduce the concentration of free calcium in the cytoplasm

calcium homeostasis: ER

• STIM1: activated if endoplasmic reticulum calcium concentration is too low

  • opens the CRAC channel

• SERCA: main way to get calcium in ER from cytoplasm

  • low free calcium in cytoplasm → moves against concentration gradient

    • needs energy from ATP

• RyR +IP3R proteins: ligand bound

  • IP3R: IP3 ligand

  • RyR: Calcium is ligand

  • moves calcium out, down its concentration gradient

calcium homeostasis: Mitochondria

• VDAC: bidirectional movement of calcium from cytoplasm to intermembrane space

• MCNX: Sodium down(into the matrix) and Calcium up(into the intermembrane space)

• MCU: calcium down into the matrix

Use of alien buffers to study calcium control of transmitter release

• alien: exogeneous

• if buffer works=decreased NT release=no postsynaptic effect

• EGTA(high affinity, slow binding): not disrupted, calcium enters close to where release occurs in terminal

• BAPTA(high affinity, fast binding): decrease in postsynaptic response because it’s fast

  • fast buffer needed shows calcium entry is near terminal

calcium entry detected by a calcium sensitive dye within the squid giant synapse during a train of action potential

molecules that exist that help to detect calcium

calcium entry is restricted to active zones of the frog nerve terminal

• pre- and post- almost directly lined up with each other

• red: high increase in calcium concentration

• calcium entry into nerve terminal isn’t uniform

  • located in discrete areas(microdomains)

• areas of calcium entry correspond with active zones

  • active zone=site of NT release; directly opposite of endplate of muscle fiber where AChRs are

GCaMP molecules

• green fluorescent protein: molecule that will fluoresce when exposed to blue light

• Calmodulin(CaM: short for calcium modulated protein): a type of calcium binding protein found in neurons and other cells

• Green fluorescent calmodulin protein(GCaMP): will fluoresce when calcium is bound to them

  • can genetically modify so it’s only expressed in neurons of your choice

GCaMP calcium indicators

• 1 Tick=1 AP

• Shows calcium spikes

Calcium is necessary for vesicle release and DURING nerve stimulation

• remove calcium from bath

• pulse calcium onto terminal

• add ionic blocker of calcium channels

• measure EPP to see if ACh was released

• EPP only seen with calcium applied during stimulus(not after stimulus, or when no calcium present)

extracellular calcium is necessary for transmitter release

no EPP with no extracellular calcium

Experimental evidence that calcium is sufficient for vesicle release

• to show sufficient, must show that just having calcium is enough for vesicle release

  • calcium liposome: vesicle with calcium; apply to cell→membrane fuses→calcium delivered to nerve terminal

  • calcium ionosphere: molecule delivered to cell that forms a channel specific for calcium(don’t need to depolarize to open like normal VG calcium channel; way to solely deliver calcium without extra shit)→EPP

  • use caged calcium

    • application of UV light breaks calcium free

    • more timely and a more accurate about of calcium in this experiment

Calcium release relationship

• not a liner relationship, rather it is exponential

• slope is usually 4 when put in log form(“fourth order relationship”)