Graeme Witton Flashcards
HCN Structure + Basic
HCN
Activated by hyperpolarisation ~-80/90mV - cAMP causes increased sensitivity (right-ward shift)
Sets Hz of firing in nociceptors - bind cAMP = faster rate of depolarisation = faster firing = more ‘painful’
Voltage-gated Na+ channel
Tetramers - encoded by the same gene with 4x polypeptide repeats (unlike K+ = 4 genes - therefore more diversity)!
6 TMD
P-loop
S4 = positive voltage-sensing domain; contains 4-6 arginine residues
HCN2 + HCN4 = cAMP sensitive - bind to CNBD (cyclic nucleotide binding domain)
HCN - Heart
HCN4 = Heart If = funny current
Chronotropic modulators:
Adrenaline = increase [cAMP] = increase heart rate
ACh = decrease [cAMP] = decrease heart rate
Ivabradine HCN2 inhibitor = chronic stable angina treatment; cause bradykinesia
ALSO - clinical trials for analgesia
Open-channel blocker = HCN4
Closed-channel blocker = HCN2
HCN - Pain
Prostaglandins generated by COZ enzymes
Bind to eiconosaid receptors = Gq - increase [cAMP]
Opioids = Gi/o - decrease [cAMP]
NSAIDs = inhibit COX
Prostoglandins also:
Bind to eiconosaid receptors
Increase PKC = reduce temperature threshold for TRPV1 activation to 35 (physiological temperature)
= thermal allodynia!!
HCN2
Found in C fibres
Polymodal - mainly heat
Unmyelinated, thinnest diameter, slowest
Global k/o = severe phenotype - ataxic, epileptic
Knock-out w/ Nav1.8 = normal pain thresholds, no heat hyperalgesia following inflammation in 3 models (good validity - carrageenan injection, formalin, prostaglandins)
BUT - Nav1.8-expressing neurones killed w/ diphtheria toxin + neuropathic pain not diminished
Unsure how HCN2 can be the root of neuropathic pain - maybe synaptic rewiring occurs in the dorsal root- function transferred to another subset of neurones? Pain = fundamental to live!
Contradictory:
PKA P @ serine residues on Nav1.8 IC loop = promotes nociceptor excitability
Nav1.8 deletion = does not reduce neuropathic hyperalgesia BUT pharmacological blockade reduces neuropathic pain
Genetic loss compensated by an up-regulation of other Nav family members, whereas no time for changes in ion channel expression following pharmacological blockade
HCN 1
Larger nociceptors - Adelta + Abeta
Nociceptors
C = polymodal; mainly heat (also: pressure + chemical)
A delta = cold
A beta = touch
Diameter/speed: A beta > A delta > C
Non-myelinated = C
P2X - Structure
P2X = non-selective cation channel - purinoreceptors P2Y = GPCR
2 TMDs
Large EC loop with cysteine residues - form disulphide bridges - holds EC domain in a structure
3x lysine positive cluster = ATP binds (not Walker domain)
Zinc = PAM (potentiates channel opening by ATP)
Trimers
- Chemical cross-linking + immunoblotting: 70kDa + 210kDa
- Atomic force microscopy = shine a laser on a flat surface, reflected at a set angle; meets a non-flat surface, angle changes = provides the architecture of the EC surface of the membrane
HA + Flag epitope tagging; co-immunoprecipitation
- P2X6 - cannot form homomers
- P2X7 - cannot form heteromers
P2X2 + P2X3 = unique properties
P2X2 homomers = meATP-insensitive, non-desensitising
P2X3 homomers = meATP-sensitive, desensitising
P2X2/3 heteromers = ATP-sensitive + non-desensitising
***cannot perform with other studies = no identified unique properties!
Concatenation - variation on 2 subunits in a trimer:
-Link with flexible, inert peptide such as glycine
= 2, 3, 3
Number of binding sites - concatenation with mutated lysine residues
= Can get 3 binding but 2 is sufficient
Is there an allosteric change occurring (cooperativity) = has not been studied!!!
P2X1-6
P2X5 = some Cl- permeability!!!
All have high Ca permeability!
Chronic stimulation - pore dilates over time - permeable to larger cations (NDMG)
Shown by a change in reversal potential over time
Controversial whether the pore dilates - whole-cell recordings are not evidence!!!
- Could be down-stream activation of a secondary pore that permits uptake of dyes/cations
- Need single-channel recordings for definitive evidence!!!!
P2X7
Chronic stimulation - large C-terminus recruits pannexin hemichannels
- Multiple permeation pathways
ie. dyes: YoPro (cationic), ethidium - Some cationic selective; some anionic selective
- Some Ca-dependent; others non-Ca-dependent
Separate pathway - pannexin-inhibition = inhibits P2X-mediated dye uptake, but not the membrane currents
Macrophages - up-regulated in DRG in chronic neuropathic pain
- Secrete beta-interleukin
- Cause a down-regulation of astrocytic EAAT1/2 = implicated in stroke/excitotoxicity
P2X7 k/o mice = reduced arthritis following collagen injection
Novel approach to deliver impermeable drugs to cells with the receptor = more specific, targeted delivery
LINK TO ANJA:
Adrenaline binds to alpha-1 receptors on astrocytes - release ATP - bind to P2X7 receptors on glutamatergic cells, cause an increase in AMPAR insertion = gliotransmitter contributes directly to the regulation of postsynaptic efficacy at glutamatergic synapses in the CNS
P2X4
Peripheral nerve injury - microglia activated, cause up-regulation of P2X4
P2X4-stimulated microglial = release BDNF
-Causes hyperexcitability via collapsing Cl- gradient
Up-regulation of P2X4 due to nerve injury damaging BBB - leakage of fibronectin
Binds to integrins on microglial = IC cascade = increase gene expression for P2X4R expression
P2X3
Found on nociceptors - up-regulated in the DR after injury
How do we know whether N-terminus/C-terminus is IC or EC?
Tag N-terminus with epitope ie. Ha, Flag
Express in a cell
Add AB to intact cell - can only bind to EC surface
Permeabilise and add AB - can bind to IC
How many binding sites are required for a fully functional channel?
Concatenation studies with mutated binding sites
P2Y
Gi/o
Microglial
Mechanism unknown - activation involves P2YR-activated microglia adhering + engulfing injured + uninjured axons (can see w/ electron microscopy)
5-HT3
Cis-cis loop receptor 4TMD (EC N + C) S2 = pore-lining EC cys-cys loop = disulfide bonds - involved in hetereodimerising EC domains = beta sheet IC domains = alpha helix
5x binding sites located at the interface of 2 subunits in an electronegative cleft
Pharmacological studies: agonists + antagonists bind at AA interface BUT atomic force microscopy: revealed a BABBA arrangement (no AA)
Pentameric - pseudosymmetric
Subunits = A, B, C, D, E
A = required to form a functional heteromer
Only 5-HT3A form functional homomers!
Weird features of 5-HT3 receptors
5-HT3a homomer = 0.4pS
5-HT3a/b heteromer = 16pS
5-HT3b cannot form functional heteromers
Thought - S2 was responsible for the conductance (lines the pore)
Chimeric DNA fusion proteins - cis loop of B responsible for high conductance
Also: crystallised 5-HT3A receptors - proteolysis experiments, cleaved IC domain = increased conductance - therefore IC domain involved in conductance/permeation
Portal holes:
A = ring of positive charge - arginine residues = smaller conductance (remove = increase conductance)
B = no arginine residues
5-HT3 drugs
Chemotherapy + severe IBS
- Agents secrete seretonin from enterochromaffin cells in the small intestine
- Vagal nerve 5-HT3 receptors = project to the medullary enteric/vomiting centre = nausea
- 5-HT3 antagonists
Epilepsy = increasing number of studies have shown that:
- Activation of 5-HT3 = inhibit epileptic seizures
- Inhibition of 5-HT3 = promote spike waves
TRPV - Structure
Vaniloid receptors = non-selective cation channel\
Highly Ca permeable
Also permeable to H+!
Same structure as a voltage-gated Na+ channel
Tetramer
6 TMDs
P-loop between S4 + S5
6x ankyrin repeats - fold to form a single, linear structure which mediates protein-protein interactions
IC capsaicin binding domain = vaniloid binding pocket
H+ = main endogenous ligand - can act as ligand or PAM (bind to EC domain)
Temperature - causes a physical conformational change + activation >43 (noxious heat) S5/S6
High voltage!
Prostaglandins
- Bind to eiconosaid receptors (Gq)
- Increase PKC
- PKC-dependent increased response in TRPV1
- Activated at lower, non-noxious, physiological temperatures ~35 = thermal allodynia
TRPV Weird
Increased permeation over time!
Different mechanism to increased permeation of P2X receptors
Channel opens at the top, closes around the molecule and pushes it through
Mutations in the pore suggest that it is the pore itself which dilates
Use to deliver membrane-impermeable local anaesthetics
= QX-314 to TRPV1-expressing cells
= agonist-dependent delivery
TRPV Drugs
Cardiovascular
TRPV2 = associated with changes in blood pressure and cardiac function
-Over-expression in the heart induces dystrophic cardiomyopathy
BUT physiological activation = improved cardiac function
TRPV2 agonist PROBENECID = cardiomyopathy treatment
In vivo mouse + pig - demonstrate a positive inotropic response
Inflammatory Pain Models
Formalin - injection 4% into hindpaw
BU: non-specific stimulus which engaged multiple pain + non-pain pathways
Prostoglandins - injection into paw
Carrageenan injection
Capsaicin injection
Complete Freud’s adjuvant (CFA)
Neuropathic Pain models
Sciatic nerve ligation
Spinal nerve ligation
Pain behaviours (rodents)
Paw licking, biting, lifting
What channel does zinc act as a positive allosteric modulator at?
P2X receptors!!
Potentiates channel opening
