L2, Sensing with channels

Question 1

Trp identification and naming:

Answer 1

• First identified in Drosophila
• Mutants found to have transient receptor potentials
• See WS1: Patch clamp technique

Question 2

Trp channel mechanistic overview: (Drosophila response to light)

Answer 2

• Rhodopsin (GPCR) senses light
• Metarhodopsin activated -> ..-> PIP2 -> IP3 + DAG
• Activation and opening of Trp channels
• Depolarisation -> Neuronal signalling
• i.e. Channels are closed in the dark, open in light -> AP

Question 3

Phototransduction in invertebrates:

Answer 3

• Light stimulates rhodopsin -> metarhodopsin
• cGMP -> 5’GMP
• Activated cNGCs (open in dark)
• Light leads to closing of channel -> hyperpolarisation

Question 4

Trp channel superfamily members:

Answer 4

• Many members
• Subdivided into TRPA, C, M, ML, P, V

Question 5

Distinguishing features of Trp channels:

Answer 5

• 6 TMS domains
• 1 pore domain -> all are cation selective (usually prefer Ca2+ over Na+)
• No voltage sensor
• Long C and N-termini for regulation of gating
• Highly promiscuous gating
• Key sites: Phosph. sites, ankyrin repeats, Calmodulin binding sites

Question 6

Regulation of Trp channels (broad categories):

Answer 6

1. Receptor activation: GPCRs and RTKs that activate PLCs modulate TRP channels activity via PIP2, DAG or IP3-mediated Ca2+ release from internal stores
2. Ligand activation: Either via exogenous small organic molecules (e.g. Capsaicin), inorganic ions and purine nucleotides
3. Direct activation: Changes in ambient temperature or mechanical stimuli

Question 7

What TRP channels are involved in thermosensation?

Answer 7

• TRPV1, V2, V3, V4, M2 -> activated by heating
• TRPM8, A1 -> activated by cooling
• Different channels concerned with different ranges of temperature -> many together confer broad range

Question 8

Evidence for TRPV1 role (mice):

Thermosensation

Answer 8

• TRPV1 deficient mice lack a subset of neurons that respond to moderate heat
• Delayed response to painful heat

Question 9

Evidence for TRPM8 role (mice):

Thermo- and chemosensation

Answer 9

• Null mutant showed no preference for comfortable vs variable temperature chambers
• Wild type prefer comfortable chamber
• Neurons for TRPM8-/- mice show loss of cold sensitivity
• Lack of flinching to acetone
• Evidence for primary role in sensing

Question 10

Mechanism for thermosensing: TRPV1

Answer 10

• Channel opening shows a much steeper temperature dependence than channel closing
• As temperature increases, channel opening is favoured relative to channel closure

Question 11

Mechanism for thermosensing: TRPM8

Answer 11

• Temperature dependence for channel opening is much less steep than that for channel closing

Question 12

Mechanosensation and hearing:

Answer 12

• Fluid filled compartment of hair cells with stereocilia on top in ear
• Vibration transmitted into inner ear, moves fluid, stereocilia jostled from side to side
• Generates receptor potential in hair cells
• Rapid mechanism; can hear at frequencies from 200 to 20,00 Hz -> msec valuesM

Question 13

Mechanotransduction in hearing:

Answer 13

• Stereocilia consists of two structure joined by tip link; movement stretches out tip link -> depolarisation -> receptor potential transmitted along to brain
• Rapid repsonse; direct effect on ion channel

Question 14

Evidence for role of TRPA1 in mechanotransduction in hearing:

Answer 14

• TRPA1 highly expressed in hair bundles
• Maturation of hair cell mechanosensitvity coincides with TRPA1 mRNA expression
• Disruption of TRPA1 in zebra fish and mice inhibits mechanotransduction
• Structure of tail contains many ankyrin repeats -> anchoring to cytoskeleton to generate force upon mechanical stimulus

Question 15

TRP targets of sensory molecules (x4):

Answer 15

• Capsaicin, piperine: TRPV1
• Menthol, eucalyptol: TRPM8

Question 16

TRPV1 in chemosensation: (Scoville relationships, species comparisons)

Answer 16

• Currents elicited by capsaicin and extracts derived from 4 different varieties of peppers
• Scovilles: Dilution factor of the spice required to be barely detectable on tongue
• Ability to sense heat aligns with scoville scale
• Birds are insensitive to capsaicin
• A tyrosine serine motif in the linker between TM2 and TM3 important for capsaicin binding; missing in chickens

Question 17

+ Sour taste detection

Answer 17

• 5 basic taste modalities in humans: Sweet, bitter, sour, salty, umami
• Detecting sourness is important in spoiled/rotten foods (acidic)
• Candidate sensor: PKD2L1
• Study involved knockout mice for PKD2L1 -> no response to sour stimuli
• Also expressed in specific neurons surrounding the central canal of the spinal chord (pH sensing -> useful for maintaining control over peripheral respiration; measuring [CO2] via pH)