Calcium Homeostasis

Question 1

Explain Calcium Homeostasis?

Answer 1

• ‘On’ reactions introduce Ca2+ into the cytoplasm
• ‘Off’ reactions remove Ca2+ from the cytoplasm
• Channels in the plasma membrane and endoplasmic/sarcoplasmic reticulum are responsible for the ‘on’ reactions
• Pumps and exchangers are responsible for the ‘off’ reactions
• Rapid, highly localised Ca2+ spikes regulate fast responses
• Repetitive, global transients control slower responses
• Non-excitable cells - GCPRs activate PKC which produces DAG or IP3
• IP3 interacts with receptor on SR and causes release of Ca2+
• Released Ca2+ activates fertilisation, cell proliferation, metabolism, and secretion
• Excitable cells - Ca2+ released by receptor or voltage gated channels in PM
• Ca2+ release causes neuron action potential, insulin exocytosis, and cardiac cell contraction

Question 2

What are the key proteins and their functions involved in Calcium Homeostasis?

Answer 2

Channels
* PM Channels = ROCs, VOCs, & MOCs

Pumps
* SERCA = SR/ER Ca2+ pump INTO ER
* PMCA = Plasma Membrane - Out of cell (ECF)

Question 3

What is the approximate Ca2+ concentration in the cytoplasm and in the ER of a non-excitable cell?

Answer 3

• Cytoplasm - 100nm
• ER - 1µM

Question 4

What is the approximate Ca2+ concentration in the extracellular fluid outside the cell?

Answer 4

1mM

Question 5

What is a calcium transient?

Answer 5

A temporary and rapid spike of Ca2+ occurs due to release and reuptake up its concentation gradient

Question 6

How is a calcium transient formed?

Answer 6

Formed through action potential which triggers opening of voltage gated Ca2+ channels or by triggered by ligand binding to ligand gated Ca2+ channels

Question 7

How is a calcium transient described?

Answer 7

Described as a rapid, temporary increase in intracellular calcium concentration, followed by a quick return to baseline levels.

Question 8

What mechanism causes the Calcium to increase in a Ca2+ transient?

Answer 8

• Ligand or voltage gated ion channels open, quickly raising cytosolic Ca2+ concentrations due to concentration gradient
  OR
• IP3 activates Ca2+ channel on SR via IP3 receptor which leads to an influx of Ca2+ ions down its concentration gradient

Question 9

What mechanism causes the Ca2+ concentration to decrease in a Ca2+ transient?

Answer 9

Ca2+ is removed back up its concentration gradient to ER, Mitochondria, or ECF by SERCA or PMCA pumps respectively

Question 10

Describe the key aspects of excitation-contraction coupling

Answer 10

• Starts with electrical excitation of heart and ends with contraction of heart muscle
• Action potential opens voltage-gated Ca2+ channels (1) increasing intracellular Ca2+ concentrations
• Ca2+ increase triggers RyR calcium release from SR (2)
• This is called a Calcium Spark
• Summation of sparks forms calcium transient which results in contraction

Question 11

What is a calcium spark?

Answer 11

RyRs bind Ca2+ and release more Ca2+ from SR

Question 12

Describe the relationship between voltage and the calcium transient in excitation-contraction coupling

Answer 12

A drop in voltage due to Na+ influx into cytosol causes activation of voltage gated Ca2+ channels to open

Question 13

Describe the absorbance and emission of Flura 2 in the presence and absence of calcium

Answer 13

Ca2+ Bound
* Absorbance = 335nm
* Emission = 510nm

Ca2+ Free
* Absorbance = 360nm
* Emission = 510nm

Question 14

What is a radiometric calcium indicator?

Answer 14

Molecules that bind Ca2+ and predictably change absorption characteristics of Ca2+ bound and unbound states

Question 15

What are the advantages to using a radiometric indicator over a standard indicator for calcium measurement?

Answer 15

• Renders the dye-concentration independent
• Unaffected by illumination intensity, probe loading, bleaching, or optical path length

Question 16

What is the emission intensity of a fluorescence molecule proportional to?

Answer 16

Emission intensity is proportional to the amplitude of the fluorescence excitation spectrum at the excitation wavelength

i.e. the greater the number of molecules at absorb a photon, the greater the number of molecules that will emit a photon as fluorescence

Question 17

Why is Fura 2 AM used in preference to Fura 2 for cell calcium measurements?

Answer 17

• Fura 2 AM has an acetoxymethyl group that makes it membrane permeable
• Esterase cleaves AM group once inside the cell

Question 18

What is a genetically encoded calcium indicator (GECI)?

Answer 18

A FRET pair attached to a protein complex that can bind and indicate the presence of Ca2+ through detection of acceptor emission

Question 19

Describe the key components in a GECI and explain how a GECI can be used to measure calcium

Answer 19

• Calmodulin and binding peptide have a donor and acceptor FP attached
• Binding of Ca2+ to calmodulin causes calmodulin to bind calmodulin-binding-peptide, and hence the donor and acceptor molecules are brought closer together
• Presence of Ca2+ can therefore detected by fluorescence emission of the acceptor

Question 20

How can a GECI be targeted to the ER?

Answer 20

• KDEL is a target peptide sequence located on the C-terminal end of AA structure of a protein
• KDEL sequence prevents a protein from being secreted from the ER and facilitates its return if accidently exported
• A plasmid can be used to transport genes into host DNA

Question 21

What are the key items in the plasmid pCMVG-CEPIA1er that results in the calcium indicator being expressed and retained in the ER?

Answer 21

• KDEL ER retention signal sequence retains protein in ER
• ER signal sequence targets protein to ER if moved to cytosol