L6, Ca-signalling toolkit I

Question 1

Basis of the calcium signalling toolkit:

Answer 1

• Stimulus
• -> Generation of calcium-mobilizing signals -> ON mechanisms
• Activation of calcium -> Calcium-sensitive processes
• Conversely, OFF mechanisms lower to resting calcium levels (ensuring correct specificity, avoiding toxic Ca2+ levels)

Question 2

Calcium signalling across kingdoms:

Answer 2

• Ubiquitous
• Can be involved in same processes across kingdoms (e.g. fertilisation in mice, sea urchins and sea weed)

Question 3

Resting vs activated levels of cytosolic free Calcium, extracellular free calcium:

Answer 3

• Resting: 100nM
• Activated: 500-1000nM
• Extracellular: ~1mM

Question 4

Calcium signalling in pancreatic acinar cells:

Answer 4

• Cholecystokinin (CCK) stimulated alpha-amylase secretion
• CCK produced in duodenum in response to hormones
• One produced, alpha amylase eventually emptied into gut -> peptide and AA digestion
• Calcium signalling event in which CCK causes an increase in [Ca2+]cyt, triggering machinery responsible for secretion of the amylase

Question 5

How is [Ca2+]cyt measured?

Answer 5

• Photoproteins e.g. aequorin, which photoluminsesces upon binding
• Fluorescent Ca2+-sensitive indicators e.g. Fura-2
• Genetically encoded calcium indicators (GECIs)…
• -> Recombinant aequorin
• -> Fluorescence resonance energy transfer (FRET) based sensors e.g. Ca2+ cameleons

Question 6

Types of fluorescent indicators:

Answer 6

• Single-wavelength indicators (e.g. Calcium green) -> fluorescence dependent on concentration
• Ratiometric indicators (e.g. Fura-2) ratio of changes -> independent of concentration of indicator

Question 7

GECIs:

Answer 7

• FRET-based
• Fluorescent proteins change orientation of CFP and YFP upon calcium binding -> differing fluorescence

Question 8

Broad mechanisms for increasing cytosolic free calcium

Answer 8

• Ca2+ entry (influx across PM)
• Ca2+ release (from I-C stores)

Question 9

5 Groups of channels that regulate calcium entry from PM

Answer 9

• Voltage operated calcium channels/VOCs (CaV; voltage-gated Ca2+ channel, TRP)
• Receptor operated calcium channels/ROCs (iGluR, P2XR)
• Store operated calcium channels/SOCs (CRAC/Orai)
• Second messenger operated channels/SMOCs (CNG)
• Ca2+ permeable mechanosensitive channels (PIEZO, CSC/OSCA)

Question 10

GPCR general structure:

Answer 10

• 7 TMS alpha helices, 4 E-C and 4 cytosolic domains
• Frequently have 8th alpha helix parallel to membrane, acting as an anchor using palmitic acid
• Acting as receptor for G proteins

Question 11

Three principal groups of GPCRs:

Answer 11

• Family A: Classic structure as above. Largest family responding to various stimuli (biological amines, light or odorants, peptides, purines, lipids etc)
• Family B: Additional large E-C domain at N-terminus for interaction with ligand (stimuli = peptides)
• Family C: Large venus flytrap domain ‘closes’ around ligand -> activation. Frequently dimerised (stimuli = biological amines, glutamate)

Question 12

How do G proteins act as molecular switches and timers:

Answer 12

• Switches: GDP bound = OFF whereas GTP bound = ON
• Timers: High GTPase activity = brief activation whereas low GTPase activity = prolonged activity