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Title: Identification and characterisation of sodium channel Nav1.7 protein-protein interactions using an epitope-tagged gene-targeted mouse
Author: Kanellopoulos, Alexandros
ISNI:       0000 0004 8499 9500
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Voltage-gated sodium channels are fundamental to the electrical properties of neurons. In mammals, ten isoforms of sodium channel are known and have been characterised (Nav1.1-1.9 and NaX). One particular subtype Nav1.7, plays a critical role in pain pathways and has a large number of functions including the control of neurotransmitter release, synaptic integration and the conduction of nociceptive signals. Humans born with loss of function mutations in this channel are unable to feel pain. This heritable disease known as congenital insensitivity to pain (CIP) can be recapitulated in transgenic mouse models and has made Nav1.7 a promising target for the development of analgesic drugs. However, this phenotype cannot be recapitulated with even the most specific of Nav1.7 channel blockers, possibly indicating that this channel's role in pain signalling is more complex than simply signal transmission. In this thesis, we aimed to gain a greater understanding of Nav1.7 and its role in pain. Firstly, to further explore the function of this channel, we used an epitope-tagged Nav1.7 mouse that showed normal pain behaviour to identify and study the proteins that interact with this channel. The high-affinity epitope-tag consisted of a HAT-tag in tandem with a 3X FLAG tag separated by a Tobacco Etch Virus (TEV) cleavage site to enable two rounds of protein purification. Nav1.7 distribution throughout the central and peripheral nervous system was determined using Tap-tagged Nav1.7 mice. A Nav1.7 complex affinity-purified under native conditions by mass spectrometry revealed over 300 proteins associated with Nav1.7 in vivo, including previously known and novel interacting proteins. We conducted both validatory and functional assessments on these proteins. Previously known interactors included β-subunits as well as other signalling and synaptic proteins. We also showed an association with the Colapsin Response Mediator Protein 2 (CRMP2) and conducted a functional validation of this protein with regards to it's trafficking role of Nav1.7. Furthermore, we identified this protein as one of the targets of the analgesic drug Lacosamide. We show that lacosamide acts through Nav1.7 in both a direct manner and through CRMP2 to reduce Nav1.7 channel current density. We also found and validated a number of synaptic proteins that interact with Nav1.7 such as Snap25 and synaptotagmin I and II. Moreover, we validated a number of interesting Nav1.7 interactions including L-type amino acid transporter 1 (Lat1), transmembrane P24 trafficking protein 10 (Tmed10), Neurofascin, A-kinase anchoring protein 12 (AKAP12) and G-protein regulated inducer of neurite outgrowth 1 (GPRIN1) (a µ-opioid receptor-binding protein), demonstrating a physical and functional link between Nav1.7 and opioid signalling. We further studied this link between Nav1.7 and opioid signalling. Type-A GPCRs are known to be regulated through a specific sodium binding site the occupancy of which diminishes agonist binding. We used an electrophysiological assay of Protein Kinase A activity to examine the role of intracellular sodium on opioid signalling. Phosphorylation of sodium channel Nav1.8 by activation of Protein Kinase A with db-cAMP is unaffected by altered intracellular sodium. By contrast, there is a dose-dependent inhibition of fentanyl action on Nav1.8 currents when intracellular sodium is increased from 0mM to 20mM. Fentanyl shows a 50% loss of activity and 80-fold increase in EC50 with 20mM intracellular sodium. These data suggest an effect of altered intracellular sodium levels on opioid receptors, where it might play a role in the modulation of opioid receptor signalling.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available