Calcium Homeostasis Flashcards
Explain Calcium Homeostasis?
- ‘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
What are the key proteins and their functions involved in Calcium Homeostasis?
Channels
* PM Channels = ROCs, VOCs, & MOCs
Pumps
* SERCA = SR/ER Ca2+ pump INTO ER
* PMCA = Plasma Membrane - Out of cell (ECF)
What is the approximate Ca2+ concentration in the cytoplasm and in the ER of a non-excitable cell?
- Cytoplasm - 100nm
- ER - 1µM
What is the approximate Ca2+ concentration in the extracellular fluid outside the cell?
1mM
What is a calcium transient?
A temporary and rapid spike of Ca2+ occurs due to release and reuptake up its concentation gradient
How is a calcium transient formed?
Formed through action potential which triggers opening of voltage gated Ca2+ channels or by triggered by ligand binding to ligand gated Ca2+ channels
How is a calcium transient described?
Described as a rapid, temporary increase in intracellular calcium concentration, followed by a quick return to baseline levels.
What mechanism causes the Calcium to increase in a Ca2+ transient?
- 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
What mechanism causes the Ca2+ concentration to decrease in a Ca2+ transient?
Ca2+ is removed back up its concentration gradient to ER, Mitochondria, or ECF by SERCA or PMCA pumps respectively
Describe the key aspects of excitation-contraction coupling
- 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
What is a calcium spark?
RyRs bind Ca2+ and release more Ca2+ from SR
Describe the relationship between voltage and the calcium transient in excitation-contraction coupling
A drop in voltage due to Na+ influx into cytosol causes activation of voltage gated Ca2+ channels to open
Describe the absorbance and emission of Flura 2 in the presence and absence of calcium
Ca2+ Bound
* Absorbance = 335nm
* Emission = 510nm
Ca2+ Free
* Absorbance = 360nm
* Emission = 510nm
What is a radiometric calcium indicator?
Molecules that bind Ca2+ and predictably change absorption characteristics of Ca2+ bound and unbound states
What are the advantages to using a radiometric indicator over a standard indicator for calcium measurement?
- Renders the dye-concentration independent
- Unaffected by illumination intensity, probe loading, bleaching, or optical path length
What is the emission intensity of a fluorescence molecule proportional to?
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
Why is Fura 2 AM used in preference to Fura 2 for cell calcium measurements?
- Fura 2 AM has an acetoxymethyl group that makes it membrane permeable
- Esterase cleaves AM group once inside the cell
What is a genetically encoded calcium indicator (GECI)?
A FRET pair attached to a protein complex that can bind and indicate the presence of Ca2+ through detection of acceptor emission
Describe the key components in a GECI and explain how a GECI can be used to measure calcium
- 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
How can a GECI be targeted to the ER?
- 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
What are the key items in the plasmid pCMVG-CEPIA1er that results in the calcium indicator being expressed and retained in the ER?
- KDEL ER retention signal sequence retains protein in ER
- ER signal sequence targets protein to ER if moved to cytosol
