VGSC and Pain

Question 1

1. what is acute pain?
2. name and describe the 2 types of chronic pain.
3. give 2 reasons as to why as a population, we all have different sensitivities to pain
4. name 2 extreme pain conditions
5. why is conditions of lack of pain sensation dangerous?

Answer 1

1. a negative emotional and physical response that alers the body to stimuli that have the potential to damage tissue
2. neuropathic pain - result of nerve damagechronic inflammatory pain - associated with chronic inflammatory disorders
3. psychological phenomena and physiological varients (such as SNPs in SCN9A)
4. primary erythermalgia and paroxial extreme pain disorder
5. because there is no inhibition of behaviour that is dangerous

Question 2

1. when is pain perceived?
2. what is the structure of DRG neurons?
3. describe the 3 stages of how a painful stimuli produces the pain sensation
4. name the 3 types of DRG fibres, their properties, and the mode of stimuli that they transmit.

Answer 2

1. when the pain signal is sent to the brain from the DRG via ascending fibres
2. bipolar - the cell body is found in the DRG, and it has terminals in the periphery and the spinal cord
3. TRANSDUCTION - receptors transduce the painful stimuli into an action potentialCONDUCTANCE - the AP is conducted to the spinal cord terminalTRANSMISSION - synaptic transmission to evoke an AP in secondary spinal cord neurons so that the signal can be conducted to the brain.
4. Aβ - large myelinated fibres; fast conductance; innoxious stimuliAδ - medium fibres that are thinly myelinated; moderate conductance that respond to painful stimuli (produce sharp pain)C - small, unmyelinated fibres with slow conductance that respond to painful stimuli (dull pain)

Question 3

1. How do NSAIDs relieve pain?
2. which patients can’t take NSAIDs?
3. where do opioids exert their action?
4. name 2 short term side effects of opiods
5. name 3 long term side effects of opoids.

Answer 3

1. inhibiting cox enzymes therefore prostaglandins can’t be produced. prostaglandins increase pain sensitivity by lowering the pain threshold
2. heart and renal condions and diabetics
3. opioid receptors in the CNS, PNS and GI
4. nausea and consitpation
5. tolerance and dependence

Question 4

VGSCs

1. what is the channel pore formed by?
2. what forms the selectivity filter and how?
3. how does the S4 domain mediate Vdep channel gating?
4. how does the dual gating of the channel enable it to respond to consecutive action potentials
5. name the 3 TTX insensitive Na_v channels

Answer 4

1. S5 and S6 and the P loop
2. the P loop. It contains negatively charged amino acids to attract positively charged ions. However, it is only wide enough for Na⁺ to pass through, thus excludes Ca and K ions
3. as the Vm changes, it moves its position. When the membrane becomes positive, it moves towards the positive EC leaflet therefore opens the channel
4. if it was only gated by voltage, the membrane would never depolarise, therefore promoting the opening of more channels. Dual gating limits the number of channels open at any one time allowing the membrane to be repolarised, thus the membrane can respond to another consecutive AP.
5. 1.5, 1.8 and 1.9

Question 5

1. what is the overall approach to studying the involvement of certain Nav channels in pain?
2. describe the behavioural analysis involved when studying pain.

Answer 5

1. gene targeting to create KO mice
2. acute pain thresholds to thermal, mechanical or pressure stimuliinflammatory pain modelsneuropathic pain models

Question 6

1. what is the expression pattern of Na_v1.3
2. how was the Na_v1.3 KO created?
3. what were the effects of Na_v1.3 KO

Answer 6

1. its expression decreases post natally but is upregulated in models of chronic pain
2. ablating part of domain 1
3. no ablation of acute pain, inflammatory pain nor neuropathic pain

Question 7

1. in which neurons is Na_v1.8 expressed
2. what were the effects of Na_v1.8 KO?
3. what is controversial about these results?

Answer 7

1. nociceptive sensory neurons (Aδ and C fibres)
2. no ablation of acute thermal and mechanosensory painablation of acute pressure painattenuated (reduction) inflammatory painno ablation of neuropathic pain
3. Na_v1.8 blockers have beneficial effects in terms of pain in KO mice

Question 8

1. where is Na_v1.9 expressed?
2. what were the effects of Na_v1.9 KO on neuropathtic and inflammatory pain?

Answer 8

1. PNS
2. attenuated inflammatory painno ablation of neuropathic pain

Question 9

Nav1.7

1. what are a) GOF and b) LOF mutations associated with?
2. where are they principally expressed?
3. what were the results of KO of Nav1.7 in Aδ and C fibres?
4. what were the effects of deleting all Aδ and C fibres. What does this suggest?
5. what were the results of KO of Nav1.7 in all sensory and sympathetic ganglia?
6. what do these findings imply?

Answer 9

1. a) extreme pain conditions b) loss of pain sensation
2. sensory and sympathetic ganglia
3. slight ablation of acute thermal pain, resistance to pressure and no ablation of mechanosensory pain. ablation of inflammatory pain. no ablation of neuropathic pain
4. same phenotype of knocking out Nav1.7 from these neurons, suggesting that these neurons dont play a role in neuropathic pain
5. ablation of neuropathic pain
6. Nav1.7 is an excellent drug target for the treatment of chronic pain conditions.

Question 10

1. describe the cre-lox tissue specific deletion method

2 what was used to delete Nav1.7 in all sensory neurons?

3. what was used to delete Nav1.7 in just nociceptive neurons?
4. what was used to delete Nav1.7 in all sensory and sympathetic neurons?
5. what control was performed to ensure that the phenotype produced was not due to the presence of Cre?

Answer 10

1. LoxP is inserted around the gene of interest by homologous recombinationthe insertion of LoxP promes the gene of interest for deletion by Cre enzymes. The gene will only be deleted in tissues that express Cre. For tissue specific deletion, a tissue specific promoter is hijacked.

One mouse strain contains the loxed gene, while a second expresses cre in a specific tissue of interest. Cross breeding of the 2 strains brings the 2 transgenes together so te loxed gene is deleted in the Cre expressing tissues.

2. the Advillin promoter (because advillin is expressed in all sensory neurons)
3. the Nav1.8 promoter
4. the Wnt1 promoter (because it is expressed in neural crest cells which give rise to the sensory and sympathetic ganglia)
5. comparisson of C57/B16 mice (mice just expressing Cre) with Nav1.8 cre mice

Question 11

1. what is responsible for hyperalgesia in response to inflammation?
2. what neurons are responsible for extreme cold pain?
3. what neurons are responsible for heat pain?
4. what neurons are responsible for inflammatory hyperalgesia?
5. what neurons are responsible for neuropathic pain?

Answer 11

1. increased trafficking of Nav1.7
2. neurons solely expressing Nav1.8
3. neurons expressing solely Nav1.7
4. neurons expressing Nav1.7 and 1.8
5. Nav1.7 expressing and sympathetic neurons

Question 12

1. In a study that identified SCN9A as the gene responsible for loss of pain conditions, who were the subjects?
2. how were the effects of identified mutations studied?
3. were the mutations found also found in control subjects?
4. what were the effects of the mutations on Nav1.7 currents?

Answer 12

1. 3 families from Northern Pakistan that contained individuals with lack of pain sensation, and control subjects from the same region
2. WT or mutated SCN9A-GFP and beta-subunit-RFP were expressed in HEK293 cells. Cells exhibiting both red and green fluorescence were subject to patch clamping.
3. no
4. reduced currents