L3, Calcium signalling intro

Question 1

Ion channels in signalling: 2 key roles

Answer 1

1. Electrical (FAST; m/sec): e.g. propagation of APs (sensory perception, motor responsives), usually in animals
2. Second messenger (SLOW;nm/sec): Transduction of readily diffusible cytosolic molecules. e.g. cytosolic Ca2+

Question 2

Key cytosolic Calcium dynamics:

Answer 2

• Calcium waves
• Calcium gradient
• Calcium oscillations or ‘sparks’

Question 3

Calcium waves overview (+ key example):

Answer 3

• e.g. post-fertilisation wave in eggs
• First observed in Medaka fish egg
• Synchronising the activation of the egg (i.e. cell cycles and other cellular events triggered simultaneously)
• Also prevents polyspermy

Question 4

Calcium gradients overview (+ key example):

Answer 4

• Associated with polarised cell growth
• e.g. pollen tubes of plant cells (tubular growth associated with tip high Calcium gradient; thought to induce exocytosis at growing tip -> further plasma membrane laid down -> local expansion

Question 5

Conventional dye for Calcium elevations

Answer 5

• Fura-2

Question 6

Calcium oscillations overview (+ key example):

Answer 6

• Many examples exist for Calcium increasing locally and transiently in response to environmental or external signal
• e.g. Transient and localised Ca2+ sparks observed in smooth muscle which are associated with excitation/contraction coupling
• It matters where in cell, dynamic event

Question 7

Key events in Calcium signals:

Answer 7

• Stimulus -> influx/efflux mechanisms
• Ca-spike
• Decoders (25,000 genes)
• Response through regulation of enzymes and structures/translation
• Feedback to influx and efflux mechanisms

Question 8

Cytosolic and intracellular Calcium levels, relevance for transport:

Answer 8

• Maintained very low
• 100-200nM at resting levels
• All cells have an inside cytosol negative membrane potential ranging from -60 to -300mV
• Always a driving force for Calcium influx into the cytosol
• Influx: Passive via ion channels
• Efflux: Active, using ATP hydrolysis or driver ion gradients

Question 9

Sources of calcium:

Answer 9

• Extracellular: calcium influx inot cytosol across PM
• Intracellular: calcium influx into cytosol from discrete compartments within the cell -> many pools ranging from ubiquitous sources like ER, nucleus, mitochondria to specific oes like vacuoles and chloroplasts in plant and fungi/SR in muscle cells

Question 10

Endomembrane calcium channels:

2 families

Answer 10

• IP3R family (3 members)
• RyR family (3 members)

Question 11

Plasma membrane calcium channels:

4 families

Answer 11

• Voltage gated Ca2+-permeable channel family (10x)
• TRP channel family (32x; some in endomembranes)
• CNGCs (6x)
• HCN (4x)

Question 12

IP3: Size and monomer organisation with function

Answer 12

• Formed as a tetramer -> HUGE
• Each monomer 2700 aa (313 kDa)
• Can also form heterotetramers -> exquisite complexity via balance of their different properties
• N-terminus forms ligand binding site
• Large central domain forms regulatory domain (Calcium, CaM, ATP binding, phosph. by PKA and TK)
• The C-terminus contains 6-TMS domains; channel pore

Question 13

Biophysical properties of IP3 receptors:

Selectivity, blocking, conductance (SC), gating

Answer 13

• Pore forming region between TMS5 and 6
• Huge size makes topology very difficult to determine
• Selectivity: P^Ca;P^K = 4.1
• Pharmacology: Blocked by heparin
• Single channel conductance: ~350 pS (large conductance, but not the highest)
• Gating requires IP3 binding; joins as tetramer -> IP3 binding affinity modulated by Calcium, ATP and kinases -> CICR link-in

Question 14

Different isoforms of IP3R (3 types and where they are mainly expressed, 2 key differences between them):

Answer 14

• 3 identified
• IP3R-1: expressed mainly in CNS
• IP3R-2: Predominantly expressed in hepatocytes and lymphocytes
• IP3R-3: Expression in cardiomyocytes
• Isoforms differ markedly in their response to IP3 (different affinities; 2 > 1 > 3)
• Markedly different responses to calcium; high Calcium concentrations fail to inhibit IP3R-3 activity (thus involved in CICR) whereas in type I, acts as an antagonist -> rapid negative feedback (therefore short, self limiting pulses as in smooth muscle)

Question 15

IP3Rs when challenged with antigen following calcium signal:

Answer 15

• Experiment carried out in lymphocytes
• Different subtypes of IP3R generate different Ca2+ signals indicating that the different sensitivities to IP3 and Calcium are physiologically relevant
• Cells expressing type II give a response similar to that observed in wild type; this receptor subtype must predominantly be involved in this cell response

Question 16

Structure function relationship of VGCCs:

Answer 16

• alpha-1 subunit: pore-forming, extracellular, linked to delta subunit by disulphide link; modulatory
• Gamma subunit in membrane; modulatory
• Beta subunit: cytosolic; important for activity

Question 17

Alpha subunit of VGCCs:

Answer 17

• 4 repeats of S4 family domain
• S4 domain: voltage sensor
• P-region; pore forming with conserved E
• Binding sites for DHP, beta-subunit, RyR (L-type VGCC)

Question 18

Biophysical properties of voltage gated calcium channels:

Answer 18

• Macroscopic currents more complex to analyse
• Activated by membrane depolarisation
• Allow E-C calcium influx across PM
• Present in all excitable cells in PM
• Highly selective for calcium

Question 19

Role of VGCCs in signal transduction:

3 key responses

Answer 19

• Detecting electrical signals, converting to Calcium signal (transducers)
• Responses include excitation-contraction coupling, exocytosis and gating ion channel activity

Question 20

Excitation-contraction coupling in skeletal muscle:

Answer 20

• L-type VGCCs sense electrical signal
• ‘puff’ of calcium -> signals RyR1 coupled with SR -> calcium floods cytosol (huge conductance)
• Regulates contraction via TnC, actin and myosin
• Regulates metabolic activity -> ATP generation for contraction event

Question 21

Exocytosis overview:

Answer 21

Occurs at synaptic junctions:
1. AP
2. Membrane depolarisation
3. VGCCs activation
4. Ca2+ elevation
5. Exocytosis and secretion of NT
6. Activation of ligand gated channels in post-synaptic junctions
7. Depolarisation fires new AP

Question 22

Gating of ion channels by VGCCs:

Answer 22

• Two key calcium channels: VGCC and BK^Ca
• BK channels allow potassium out of cell -> voltage gradient for VGCC to allow Ca2+ influx -> counteracting contraction of smooth muscle
• Increase BK: Vasodilation
• Decrease BK: Vasoconstriction