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!
