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Molecular Signalling > Amanda Mackenzie > Flashcards

Amanda Mackenzie Flashcards

1
Q

Roles of Ca2+ in the body

A 
Muscle contraction
Neurotransmitter release
Action potenitals
Immune cell activation
Exocytosis
Gene transcription
Memory formation
Detecting/sensory perception
Death (from too much Ca2+)
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2
Q

[Ca2+] outside cells

A

1.2 mM

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3
Q

[Ca2+] inside cells

A

50-100 nM (depends on cell)

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4
Q

Fura-2

A

Fluorescent Ca2+-sensitive indicator

Impermeable to cells

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5
Q

Fura-2/AM

A

Membrane-permeable version of Fura-2

Cleaved by esterases in the cell to regenerate Fura-2 as the free acid

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6
Q

Mechanism of Fura-2

A

Binding to Ca2+ changes its fluorescent properties
Fluorescence is measured at 340/380 and 500 nm
[Ca2+] can be calculated based on the ratios of fluorescence at these wavelengths

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7
Q

TRPs

A

Transient receptor potentials

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8
Q

Basic structure of thermo-TRPs

A

4 subunits, each with 6 transmembrane domains and cytosolic N- and C-terminal domains

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9
Q

Thermo-TRPs

A

Activated by temperature
A “thermally sensitive” subclass of TRP channels
6 have been identified so far

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10
Q

Where are thermo-TRPs expressed?

A

Primary sensory nerve terminals

Nociceptive nerve axons are either small-diameter, unmyelinated C-fibres or medium-diameter, myelinated Adelta fibres

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11
Q

TRPV1 activation

A

Activated by painful levels of heat

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12
Q

TRPV2 activation

A

Activated by painful levels of heat

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13
Q

TRPV3 activation

A

Responds to non-painful warmth

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14
Q

TRPV4 activation

A

Responds to non-painful warmth

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15
Q

TRPM8 activation

A

Activated by non-painful cool temperatures

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16
Q

TRPA1 activation

A

Activated by painful cold

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17
Q

What are the thermal thresholds of thermo-TRPs modulated by?

A

Extracellular mediators released as a result of tissue damage/inflammation e.g. bradykinin, prostaglandin, growth factors

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18
Q

TRPM2, TRPM4, TRPM5

A

Also show temperature sensitivity but not usually included in the thermo-TRP family because they are not expressed in primary somatosensory neurones

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19
Q

Therapeutic use of thermo-TRP blockers

A

Novel analgesics
They are nociceptors
Nociceptors respond to noxious mechanical, chemical or thermal stimuli through generating action potentials that are propagated to higher brain centres via the dorsal horn of the spinal cord, resulting in a sensation of pain

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20
Q

TRPV1

A

Mainly found in small to medium diameter neurones of primary sensory ganglia
Expressed by all major classes of nociceptive neurone
Only TRP family member to be activated by vanilloids (e.g. capsaicin in hot chilli peppers)
Promiscuous - activated by low pH, a wide range of agonists and by physical factors e.g. heat, membrane depolarisation
Thus, TRPV1 is viewed as a “signalling integrator” for many noxious stimuli

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21
Q

TRPV1 activation by temperature

A

TRPV1 is directly gated by noxious heat
Activation threshold >43 degrees
But threshold can be lowered by pro-inflammatory mediators released during tissue injury/inflammation

22
Q

TRPV1 activation by low pH

A

TRPV1 is directly gated by acidic conditions (pH < 6)

Low pH also sensitises TRPV1 to capsaicin and heat

23
Q

Compound that activates TRPV1

A

Resiniferatoxin

24
Q

Compound that inhibits TRPV1

A

Capsazepine

25
Q

Method used for TRPV1 cloning

A

Caterina et al. used an expression cloning strategy based on Ca2+ influx in order to isolate a functional cDNA from sensory neurones that would encode the capsaicin receptor

  1. Constructed a cDNA library from DRG-derived mRNA
  2. Subdivided cDNA library into groups of ~16000 clones and each group was transiently transfected into HEK293 cells
  3. Transfected cells were then loaded with Fura-2 and microscopically examined for changes in intracellular Ca2+ levels
  4. A positive pool was identified, subdivided and re-assayed
    In this way, an individual clone was obtained that contained a cDNA insert that conferred capsaicin and resiniferatoxin sensitivity to HEK293 cells
26
Q

Basis for method used to clone TRPV1

A

Strategy was devised based on the ability of capsaicin to trigger Ca2+ influx into sensory neurones
i.e. a non-neuronal cell containing the capsaicin receptor cDNA would have the same ability to undergo an increase in intracellular Ca2+ upon capsaicin exposure

27
Q

Capsaicin

A

Excitatory neurotoxin
Selectively destroys primary afferent nociceptive neurones in vitro and in vivo
HEK293 cells transfected with capsaicin receptor and continuously exposed to capsaicin died within several hours from excessive ion influx
Showing that capsaicin receptor expression in a non-neuronal context can repeat the cytotoxicity observed in sensory neurones

28
Q

How do many inflammatory mediators enhance heat pain during tissue injury/inflammation?

A

Sensitise the heat-gated TRPV1 ion channel through intracellular signalling pathways whose end-point is phosphorylation of TRPV1 by kinases
The mediators bind to GPCRs and cause downstream activation of PKA/PKC (Ser/Thr kinases) which phosphorylate TRPV1
This modulates TRPV1’s thermal threshold and influences its gating by a wide range of agonists

29
Q

Sensitisation of TRPV1 by NGF

A
  1. NGF acts on TrkA receptor
  2. This activates a downstream signalling pathway in which PI3K plays a crucial role
  3. Src kinase is the downstream element that binds to and phosphorylates TRPV1 at a single Tyr residue (Y200)
  4. Phosphorylated TRPV1 is then inserted into the membrane
30
Q

Effect of NGF on surface membrane expression of TRPV1

A

Enhances surface membrane expression of TRPV1 (caused substantial increase in current after saturating concs of capsaicin applied)
This effect was absent in cells with dnSrc or with TRPV1 Y200F mutants

31
Q

TRPM8

A

Also a Ca2+-permeable cation channel
Responds to non-noxious cool temperatures
Expressed in small-diameter sensory neurones lacking nociceptive markers e.g. TRPV1
Activated by menthol - produces a sensation of pleasant cooling - and other chemicals that mimic cooling

32
Q

In vivo studies of TRPM8-/- mice

A

Mice showed deficiencies in the sensation of non-noxious cool temperatures
But not deficient in response to noxious cold, showing other receptors must exist to detect more extreme cold

33
Q

Characterisation of TRPM8

A

TRPM8 was characterised and cloned from trigeminal sensory neurones

34
Q

Method used for TRPM8 cloning

A

A screening strategy based on Ca2+ imaging

  1. Constructed a cDNA library from trigeminal sensory neurone mRNA
  2. Subdivided cDNA library into groups of ~10000 clones and each group was transiently transfected into HEK293 cells
  3. Transfected cells were then loaded with Fura-2 and microscopically examined for changes in intracellular Ca2+ levels
  4. A positive pool was identified, subdivided and re-assayed
    In this way, they identified a single cDNA that conferred menthol sensitivity to transfected cells
35
Q

Why were trigeminal neurones used for TRPM8 cloning?

A

Calcium responses are more prevalent in trigeminal cultures

36
Q

Difference between TRPM8+/+ and TRPM8-/- mice

A

TRPM8+/+ mice respond to cold (18 oC), menthol and capsaicin

TRPM8-/- mice only respond to capsaicin

37
Q

How do inflammatory mediators inhibit TRPM8 in sensory neurones?

A

Inflammatory mediators e.g. bradykinin, histamine do not inhibit TRPM8 through conventional signalling patwhays
Instead, Gaq binds directly to TRPM8 and, when activated by a Gq-coupled GPCR, directly inhibits TRPM8 ion channel activity
Deletion of Gaq largely abolishes inhibition of TRPM8

38
Q

Perfusion of an “inflammatory soup” with cold-sensitive corneal nerve fibres

A

Inhibited the cold response of the neurones but enhanced the heat response

39
Q

What does the “inflammatory soup” contain?

A

Histamine and 5-HT

40
Q

How was it determined whether a diffusible intracellular mediator was involved in the inhibition of TRPM8 by bradykinin?

A

Constructed cell-attached patch recordings of single TRPM8 channels with bradykinin only applied outside the patch

41
Q

Cell-attached recording of HEK293 cells expressing TRPM8 and B2R

A

TRPM8 channel activity not inhibited by bath application of bradykinin

42
Q

Cell-attached recoding of HEK293 cells expressing TRPV1 and B2R

A

TRPV1 channel activity enhanced by bath application of bradykinin

43
Q

Conclusions from cell-attached recordings of TRPM8 channel activity upon bath application of bradykinin

A

Bradykinin-induced inhibition of TRPM8 is membrane-delimited and depends on local events within the patch, not on diffusible messengers
i.e. activated Gaq inhibits TRPM8 independently of the PLC pathway

44
Q

How was “activated Gaq inhibits TRPM8 independently of the PLC pathway” investigated?

A

Measured inward and outward currents activated by menthol from HEK293 cells expressing TRPM8 and different Gaq mutants

45
Q

Effect of overexpression of Gaq on TRPM8 inward current

A

Small inhibitory effect

Likely because a small proportion of Gaq is in the active GTP-bound form even in the absence of GPCR stimulation

46
Q

Effect of Gaq Q209L mutant on TRPM8 currents

A

Q209L mutant is deficient in intrinsic GTPase activity so is mainly in the GTP-bound active configuration
This causes a much greater inhibition of both inward and outward TRPM8 currents

47
Q

How did the investigators determine that Gaq directly inhibits TRPM8, independently of the downstream PLC pathway?

A

Constructed a chimera between Gaq and Gai2 = 3Gaqiq (B2R couples to Gq and Gi)
Replaced the PLC-bindng region on Gaq with the corresponding region on Gai
Found that the chimera completely failed to deplete PIP2 (even when containing the constitutively active Q209L mutation), but still strongly inhibited both inward and outward TRPM8 currents

48
Q

Triple mutant

A

Q209L/R256A/T257A
Chosen because it had been reported to be unable to activate PLC
Was shown to profoundly suppress TRPM8 currents, but it was also found that this mutant caused Tubby translocation so in fact does couple to PLCb
So wasn’t used (used chimera instead)

49
Q

Immunoprecipitation experiments

A

Carried out to detect protein-protein interactions

TRPM8 directly interacts with Gaq subunits in HEK293

50
Q

Tubby

A

Sensitive reporter of membrane PIP2 levels

Used to monitor activation of PLCb/PIP2 pathway

Molecular Signalling (6 decks)

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