lecture 3: CRAC

what is the activation of mast cells (and other immune cells) dependent on?

the Ca²⁺ influx from a channel called CRAC

what is Ca²⁺ required for?

• degranulation and secretion of histamines and proteases

• secretion of TNF-alpha

• synthesis and release of leukotrienes and prostaglandins

• synthesis of cytokines

what is CRAC sensitive to?

Ca²⁺ depletion in the ER

SCID patients have…

impaired T-cell function

what did using a fluorescent Ca²⁺ indicator on SCID patient cells show?

after the depletion of intracellular Ca²⁺ stores, there was a failure in the influx of Ca²⁺ into the cell

the CRAC channels in SCID patients were…

non-functional

why was screening done in fruit fly cells?

their immune cells have similar properties to those in mammals

how did scientists find out which genes are required for Ca²⁺ entry

they performed a massive genetic screen using siRNA to mute genes 1 by 1

which gene completely stopped Ca²⁺ entry

STIM1

what happened when STIM1 was silenced

the Ca²⁺ spike was almost completely eliminated, proving STIM1 is important for CRAC activation

structure of STIM1

• 1 transmembrane domain → anchors the protein into the ER membrane

• EF-hand motifs → when Ca²⁺- bound, it keeps STIM1 inactive

• 3 coiled-coil domains → involved in protein interactions

how was STIM1 translocation discovered?

using GFP, the mechanism of STIM1 was tracked

when analysing SCID patients and their family who does not have SCID, what was 2 things that they found?

• SCID is an autosomal recessive disorder

• there was a mutation in the Orai1 gene

NFAT was used to track influx, what happened when either STIM1 and Orai1 were knocked out?

NFAT did not migrate from the cytosol to the nucleus

what kind of protein does Orai1 code for?

a protein with 4 transmembrane domains, similar to CRAC

the STIM1 and Orai1 mechanism

1. when Ca²⁺ is depleting from the ER lumen, Ca²⁺ dissociates from STIM1, causing it to change shape and cluster together in the ER

2. STIM1 moves via the coiled-coil interactions until it reaches the plasma membrane

3. the cytoplasmic tail of STIM1 extends outwards, exposing its SOAR/CAD domain

4. when Orai1 encounters STIM1 at the ER-PM junction, the SOAR/CAD domain binds to the cytosoloic c-terminus of Orai1

5. the binding opens the central pore of the Orai1 channel allowing for Ca²⁺ influx

6. as Ca²⁺ refills in the ER, Ca²⁺ re-binds to STIM1, causing it to release Orai1

what happened when Orai1 was knocked out of mice?

the mice showed stunted growth (Orai1 is in every cell of the body but there are other variants so the animal will survive)

why was a blue dye injected into the ears of mice when conducting animal models?

blue dye could be taken up by mast cells. By inducing an allergic reaction, the blue dye releases

what were the results when KO mice were injected with blue dye?

the blue dye were stuck in the mast cells