Calcium signalling and store operated Ca2+ channels Flashcards
What are the roles of STIM1 and Orai1 proteins in CRAC channel function? How the distribution of STIM1 and Orai1 change upon depletion of intracellular Ca2+ stores?
In cells with full calcium stores in EF-hand calcium binding motif of STIM1 is occupied by calcium, and STIM1 is homogenously distributed in the ER. Orai 1 is dispersed on the plasma membrane. When the calcium content of the stores fall calcium dissociates from the EF-hand and STIM1 migrates to ER-plasma membrane junctions. Interaction between carboxyl termini of orai1 and CRAC activation domain of STIM1 leads to CRAC channels opening and calcium entry.
Describe the sequence of events in Gq/11 signalling pathway that precede re-distribution of STIM1 to junctional ER?
The Gq/11 protein works by activating phospholipase C (PLC). PLC then cleaves a phospholipid. In the process, phosphatidylinositol 4,5-bisphosphate (PIP2) is cleaved into diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. IP3 then diffuses through the cytosol to bind to IP3 receptors, particular calcium channels in the endoplasmic reticulum (ER). These channels are specific to calcium and only allow the passage of calcium to move through. This causes the cytosolic concentration of calcium to increase, causing a cascade of intracellular changes and activity.
What is the role of 1,4,5-inositoltrisphospate (IP3) in activation of store-operated Ca2+ channels?
IP3s main function is to mobilize calcium from storage organelles and to regulate cell proliferation and other cellular reactions that require free calcium. It indirectly activates SOC channels via the depletion of ER calcium.
Activity of which Ca2+ selective channels, CRAC or L-type, results in elevation of cytoplasmic Ca2+ concentration at a membrane potential of -60 mV?
Neither, CRAC are activated at all currents but rely on calcium concentrations and L-type need -40 to -20 to activated. The T-type are the channels that require -60mV.
Describe the mechanism of Ca2+ dependent inactivation of CRAC channels.
While CRAC are not voltage gated they will inactivate at negative potentials. Inactivate depends on calcium buffering in cytoplasm and Ca concentration in the bath.
What are the main sources of cytoplasmic Ca2+?
What is the “Ca2+ specificity problem”? How this problem is resolved?
Calcium is also known as the universal messenger, this means that it is used in almost all stimuli in cells whether they be physical or chemical involve calcium in some way. As such due to such a large amount of calcium usage it faces a problem in specificity. There needs to be distinct differences in channels and signals while still allowing to be in charge. There are only theories to understand this predicament.
- Everything is activated
- Different cells perform different task
- Calcium signals are complex (Ca2+ oscillations); they are restricted to specific sub-cellular regions and vary in amplitude and frequency (shape/kinetics).
What determines the kinetics of Ca2+ signal?
Distribution and concerted action of calcium channels, calcium pumps, transporters and buffers determine the shape (kinetics) of the calcium signal.
Which signalling pathway is activated by Gq/11? Describe the sequence of events.
The Gq/11 protein works by activating phospholipase C (PLC). PLC then cleaves a phospholipid. In the process, phosphatidylinositol 4,5-bisphosphate (PIP2) is cleaved into diacyl glycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG remains bound to the membrane, and IP3 is released as a soluble structure into the cytosol. IP3 then diffuses through the cytosol to bind to IP3 receptors, particular calcium channels in the endoplasmic reticulum (ER). These channels are specific to calcium and only allow the passage of calcium to move through. This causes the cytosolic concentration of calcium to increase, causing a cascade of intracellular changes and activity.
Which of the following channels will require longer time to activate in response to a depolarising step to 0 mV: voltage-gated Na+ channel, voltage-gated K+ channel, or CRAC channel? (trick question)
The question is wrong.
If you block SERCA pump with thapsigargin, how this will affect cytoplasmic Ca2+ concentration and why?
SERCA ensures that cyctoplasmic calcium concentration is reduced and calcium is stored in the ER. If you block SERCA pumps then cytoplasmic concentration of calcium will rise significantly because the ER is unable to pump and store it inside.
List examples of physiological functions regulated by Ca2+. Why Ca2+ is often called “life and death signal”?
Sperm motility and fertilisation
Exocytosis
Muscle contraction
Enzyme activity
Cell motility
Gene transcription
Cell growth and proliferation
It’s called the life and death signal because when calcium increases substantially it can cause necrosis but is also largely important in apoptosis. As such it can cause either life or death.
What type of Ca2+ response is activated by physiological concentrations of Cholecystokinin (10pM) in pancreatic acinar cells? How this response changes if concentration of Cholecystokinin is increased to 10 nM?
Calcium oscillations occur when 10pM CCK are present. This initiates enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, trypsin is activated while still in the cell. With 10nM of CCK there is an immediate elevation of intracellular calcium followed by a prolonged plateau phase. After a delay of approx 300 seconds there is a rise in trypsin activity to an elevated plateau level. Addition of 10 uM benzamidine, a cell-permeable trypsin inhibitor, reduces trypsin activity.
Name two types of highly selective Ca2+ channels. Give an example of non-selective Ca2+ permeable channel.
Selective:
Voltaged-gated calcium channels - excitable cells (nerve cells, muscle cells)
Store operated calcium channels - non excitable cells (immune cells, hepatocytes, etc) and some excitable cells (muscle)
Non selective:
Ligand gated channels, mechanosensitive channels, temperature sensitive channels - non excitable cells and excitable cells.
What are the main functions of CRAC channels? What known human diseases are caused by abnormalities in CRAC channels function?
Store refilling
Calcium signal amplification
ATP production
Exocytosis
Cell proliferation.
Autoimmune diseases. The activation of T lymphocytes can essentially cease to function and as such people with this type of disease cannot be exposed to any type of bacteria or fungi, virus etc. This disease is called severe combined immunodeficiency SCID, or ‘bubble boy disease’.
