Role of Ca2+ channels in cell signalling Flashcards
What are the 2 broad types of cell signalling mechanisms?
Electrical
= e.g. propagation of action potential
Second Messenger
= e.g. transduction of signals by ready diffusible cytosolic molecules = cytosolic Ca2+
What are the 3 cytosolic Ca2+ dynamics? (+examples)
Ca2+ waves
= e.g. medaka fish eggs - synchronising activation of egg
Ca2+ gradient
= e.g. pollen tubes - tip high Ca2+ gradient to induce exocytosis in growing tip
Ca2+ oscillations / sparks
= e.g. smooth muscle - excitation / contraction coupling
What are Ca2+ signals the result of?
Two opposing functions:
influx into
+
efflux out of
(the cytosol)
What are the energetics of Ca2+ transport?
cytosolic Ca2+ = maintained at approx. 100-200 nM at resting levels
inside cytosol of cell = negative membrane potential = -60 - -300 mV
= electrochemical potential gradient needed for Ca2+ influx into cytosol
= ALWAYS a driving force favouring Ca2+ INFLUX into cytosol
What are the most important aspects of Ca2+ transport energetics?
INFLUX
= energetically downhill process
= ALWAYS via ion channels
EFFLUX
= energetically uphill process
= energised by ATP hydrolysis (pumps) or driver ion gradients (carriers)
What are the 2 pools of Ca2+ outside the cytosol?
Extracellular
= Ca2+ influx into cytosol across the plasma membrane
Intracellular
=. Ca2+ influx into cytosol from discrete compartments within the cell
What are some examples of intracellular pools of Ca2+? (where are they found)
ER (ubiquitous)
SR (muscle cells)
Nucleus (ubiquitous)
Vacuoles (plants and fungi)
Chloroplasts (plants)
Mitochondria (ubiquitous)
What are some examples of Ca2+ permeable channel families in animal cells?
Endomembranes
= IP3R (inositol triphosphate receptor) - 3 members
= RyR (ryanodine receptor) - 3 members
Plasma membranes
= Voltage-gated Ca2+ permeable channel family - 10 members
= TRP - 32 members
= CNGCs - 6 members
= HCN - 4 members
What is the structure / function and monomer organisation of IP3R (3)
Formed as a tetramer (4 monomers), total size over 1 MDa
(1) N-terminus forms ligand binding site
(2) Large central domain forms regulatory domain
= Ca2+, CAM, ATP binding
= phosphorylation by PKA and TK
(3) C-terminus contains 6 TMS domains
= channel pore
What are the biophysical properties of IP3 receptors?
Selectivity
= PCa: PK = 4.1
= the pore region formed between TMS 5 + 6
Pharmacology
= blocked by heparin
Single channel conductance
= 350 pS (can allow a lot of Ca2+ in at once)
Gating
= IP3-dependent
= IP3 binding affinity (+sensitivity of channel activation) is modulated by: Ca2+, ATP + kinases
What are the 3 isoforms of IP3R, where are they expressed?
How do they differ?
IP3R-1
= CNS
IP3R-2
= hepatocytes + lymphocytes
IP3R-3
= cardiomyocytes
= differ in their response to IP3
What is the result of different responses to IP3 of IP3R isoforms?
High Ca2+ = does NOT inhibit IP3R activity
= CICR (calcium induced calcium release)
Low Ca2+ = acts as antagonist = mechanism for rapid negative feedback = short, self limiting pulses of Ca2+ release
What do different Ca2+ signals generated by different subtypes of IP3R indicate?
That different sensitives to IP3 and Ca2+ are physiologically relevant.
Cells expressing type 2 = have similar response in WT = suggests this subtype predominantly involved in cell response
e.g. study using different IP3Rs to generate different Ca2+ signatures in lymphocytes challenged with antigen
What is the structure function relationship of voltage gated Ca2+ channels (VGCCs)?
alpha subunit
= 175 kDa
= main part forming channel pore
alpha-2 subunit
= 143 kDa
= extracellular and linked by disulphide linked to delta subunit
delta subunit
= 27 kDa
= fixed in membrane by one TMS domain
gamma subunit
= 32 kDa
= 4 TMS
beta subunit
= 52-78 kDa
= cytosolic
= elevates size of current in pore forming aplha subunit
Give more detail about the structure of the alpha-subunit?
4 repeats of S4 family
S4 domain = voltage sensor
P-region = pore forming with conserved E
binding site for:
DHP, B-subunit, RyR
What are the biophysical properties of voltage-gated calcium channels?
Activated by membrane depolarisation
Allow extracellualr Ca2+ influx across plasma membrane
Present in all EXCITABLE CELLS in the plasma membrane
Highly selective for Ca2+
How do VGCCs act as signal transducers?
They convert electrical signals to Ca2+ signals.
Action Potential
Membrane Depolarisation
VGCC activation
Ca2+ influx
Elevation of cytosolic Ca2+
Downstream effect (response)
What are the 3 most researched ‘responses’ from Ca2+ elevations?
Excitation - contraction coupling
Exocytosis - e.g. releasing neurotransmitter across synaptic gap
Gating ion channel activity
What is an example of signal amplification?
Excitation-contraction coupling in skeletal muscle
What are the (7) steps of Exocytosis at synaptic junctions?
(1) Action potential
(2) Membrane depolarisation
(3) VGCCs activation
(4) Ca2+ elevation
(5) Exocytosis and secretion of neurotransmitter
(6) Activation of ligand gated channels in post-synaptic membrane
(7) Depolarisation fires a new action potential
What is an example of gating of ion channels?
Regulation of smooth muscle tone by BKCa channels
= controls vasoconstriction / dilation = blood pressure
What are the 4 main isoforms of VGCCs (+where are they expressed)?
L-type / CaV1
= skeletal , cardiac
= EC - coupling
= sensitive to DHP
= 25 pS
N-type / CaV2
= neurons, endocrine cells
= neurotransmitter release at synaptic gaps
= sensitive to conotoxin
= 13 pS
P-type / CaV2
= purkinje cells
= sensitive to FTX (funnel spider toxin)
= 10-20 pS
T-type / CaV3
= heart cells
= single channel conductance tiny
= sensitive to mibefradil
How do the different VGCC isoforms differ?
Have different sensitivities to membrane depolarisation.
e.g. T type (blue)
= LOW threshold for activation
= Ipeak = +0 mV
= activation potential = -50 mV
e.g. L, P , N types (red)
= HIGH threshold for activity
= Ipeak = +30 mV
= activation potential = -20 mV
What is a summary of VGCCs
Have different isoforms with different biophysical properties
= different single channel conductances
= different sensitivities to membrane voltage / depolarisations
Have different expression patterns
Modulation of the alpha pore subunit by auxiliary subunits (beta, gamma , alpha2delta)
What is a summary for IP3Rs?
Have different modulation of channel activity by Ca2+, ATP + phosphorylation
Have different isoforms with different biophysical properties
Have different expression of IP3R isoforms in different cell types (abundance and type)
Can be formed from heterotetramers
= different combinations of types = diversity
