2 - molecular basis of pain sensation: TRP channels

hyperalgesia

increased pain from stimulus that normally provokes pain

inflammatory

pain that occurs when tissue is damaged

• coordinated response to help tissue heal

nociceptive pain

arises from actual or threatened damage to non-neural tissue

• due to activation of nociceptive

allodynia

due to stimulus that does not normally provoke pain

• ex.) light feather touch causing pain

neuropathic pain

caused by lesion or disease of the somatosensory NS

nociception

neural process of encoding noxious stimuli

nociceptive neuron

central or peripheral neuron of somatosensory NS that is capable of encoding noxious stimuli

nociceptive stimulus

actually or potentially tissue-damaging event transduced and encoded by nociceptors

nociceptor

high-threshold sensory receptor (i.e. an ion channel) of the peripheral somatosensory nervous system that can transduce and encode noxious stimuli

noxious stimuli

stimulus that is damaging or threatens damage to normal tissues

pain pathway steps review

1. noxious stimuli convert into electrical signals by nociceptors

2. activation of ion channels alter resting membrane potention

3. depolarization evokes AP

large drg neuron

diameter = large

myelination = heavy

fiber = Aβ

conduction speed = very fast

soma size = large

medium drg neuron

diameter = medium

myelination = moderate

fiber = Aδ (delta)

conduction speed = fast

soma size = medium

small drg neuron

diameter = small

myelination = none

fiber = C

conduction speed = slow

soma size = small

current clamp

records changes in ion channel conductance

Ohm’s law

V = I R

depolarization makes membrane porentials more ______

positive

whole-cell voltage clamp

measures total ion currents across an entire cell membrane by using a glass micropipette to "clamp" the membrane potential at a set voltage

selectivity filter

narrowest point permeating ions pass through

gated ion channels

open + close depending on type

• voltage, ligand and mechanical

TRP channels

mediate sensation of pain, temperature and chemical irritants

• in drg

TRPA1

detector for allyl isothiocynate (wasabi) and is pain detector

TRPM8

mint

TRPV3

camphor

TRPV1

detector for capsaicin (chili) and noxious heat sensory

polymodal peptidergic receptors

express multiple pain-responsive ion channels

TRP channel step 1

agonists for TRP channels that cause pain activate TRPA1 channels

TRP channel step 2

compounds including bradykinin are released

• binds to BDKRB2 receptor (GPCR)

• activates TRP channels

TRP channel step 3

activation of TRP channels allow influx of Ca2+ to depolarize the cell

AP to spinal cord step 4

when terminal reaches above threshold, nociceptive neurons initiate APs

• propogate along C and Aδ fibers

AP to spinal cord step 5

pain fibers found in laminae 1 and 2 of dorsal horn

AP to spinal cord step 6

nerve terminals release neurotransmitters

• glutamate, substance P and calcitonin gene related peptide

• activates postsynaptic receptors spinothalamic tract neurons

isothiocyanates

natural compounds found in horseradish and wasabi that activate the TRPA1 channel

• also found in broccoli, kale and cababge

how do isothiocyanates bind to TRPA1

covalently binds to sulfur atom of the cytesine residue in TRPA1 to form a disulfide bond

• modified cytesines open TRPA1

• depolarization initiates AP

non-selective cation channels

callow cations such as Na+, K+ and Ca2+

• ex.) TRP channels

fluorescent calcium indicators

used to look at TRP channel activity

• measure intracellular Ca2+ concentrations

fura-2

chemical calcium indicator

• Ca2+ free and Ca2+ bound have 2 different absorption properties that can be used to quantify Ca2+ change

GCaMP

genetically encodable Ca2+ indicator

• “split” GFP molecule that is brought together by Ca2+ binding domains

CaM

calcium binding domain of calmodulin

M13

calmodulin binding domain of skeletal muscle myosin light chain

cameleon

also GECI and Ca2+ brinsg together 2 different fluorescent proteins to allow fluorescent energy transfer (FRET)

ratiometric Ca2+ indicators

measure Ca2+ by emitting light at 2 different wavelengths

negatively charged compounds are ______

cell impermeant and require invasive loading techniques

acetoxymethyl (AM) ester modification

masks negatively charged caroxylic groups and produces uncharged hydrophobic molecules that can be passively loaded into live cells

Fure2-AM

cell permeable and distributes evenly when it enters the cell

• cellular esterases remove the acetoxymethyl group once in the cell and allow Fura2 to chelate calcium

Ca2+ free Fura2 AM imaging

excitation max at ~380nm and emits 510nm

Ca2+ bound Fura2 AM imaging

excitation max at ~340nm and emits 510nm