Calcium signalling toolkit

Question 1

Free calcium

Answer 1

Extracellular calcium is 1mM
Resting cytosolic free calcium is 100 nM
Activated calcium is 500 to 1000nM

Question 2

Pancreatic acinar cells

Answer 2

Cholecystokinin (CCK) produced in duodenum in response to peptides in gut
Stimulates production of alpha amylase in pancreatic acinar cells
CCK causes an increase in cytosolic calcium, triggering machinery for alpha-amylase secretion

Question 3

Measuring calcium concentration

Answer 3

Photoproteins (Aequorin)
Fluorescent Ca2+ indicators (radiometric Fura-2)
Genetically encoded calcium indicators (GECIs). Ca2+ chameleons

Question 4

On mechanism for Ca2+ increase

Answer 4

Ca2+ entry via influx across plasma membrane

Ca2+ release from intracellular stores (mitochondria, nuclei, chloroplasts, vacuole)

Question 5

Plasma membrane calcium channels

Answer 5

Voltage operated calcium channels (VOCs) gated by membrane potential
Receptor operated (ROCs) directly perceive a stimulus (nicotine Ach receptor)
Store operated (SOCs) gated by level of calcium in endomembrane stores
Second messenger operated (SMOCs). Cyclic AMP

Question 6

Intracellular store release

Answer 6

Phospholipase C (PLC) inositol (1,4,5) triphosphate (IP3) pathway

Starts with perception of stimulus by GPCR

Question 7

GPCRs

Answer 7

7 membrane spanning alpha helices
4 extracellular domains and 4 cytosolic domains (interact with G proteins)

3 families (A,B,C)
B/C have large extracellular domains

Question 8

Heterotrimeric G proteins

Answer 8

Inactive when GDP bound to alpha subunit (guanyl nucleotide binding site)
Stimulus binding to GPCR allows interaction with alpha subunit. Exchange of GDP for GTP
Alpha subunit dissociates from beta-gamma complex and interacts with downstream effector
Intrinsic GTPase hydrolyses back to GDP

Question 9

Molecular G protein timers

Answer 9

G proteins hydrolyse GTP at different rates
High GTPase activity leads to brief activation
Low GTPase activity prolonged activation

Question 10

G protein activation in Dictyostelium discoideum

Answer 10

Monitored by FRET (fluorescence resonance energy transfer)

Cyan fl. protein attached to alpha subunit. Yellow protein to Beta-gamma complex.
Yellow fl. emitted when heterodimer is intact
Cyan fl. emitted when stimulated (alpha subunit dissociated)