PhD Studentship: Elucidating the mechanisms of regulation of Nav1.7 channel in the plasma membrane

Sensory neurons detect and transmit painful stimuli to the CNS. Inflammation and nerve injury sensitise sensory neurons which results in a decrease of pain thresholds. This can be due, at least in part, to an enhanced trafficking of voltage gated sodium channels to the plasma membrane which would result in increased excitability of sensory neurons. The VGSC subunit Nav1.7 has been shown to be crucial for pain signalling in mouse and human. Three genetic pain disorders have been mapped to Nav1.7 in humans; these are primary erthromyalgia, familial rectal pain and complete insensitivity to pain. However, little is known about how the Nav1.7 surface pool is regulated to set pain thresholds and respond to changes in the environment (e.g. inflammation). Nav1.7 surface pool is determined by mechanisms controlling its transport to nerve terminals, insertion into and endocytosis from the membrane. Investigation of these processes may lead to new druggable targets for pain relief. The aim of this project is to identify the contribution of the intracellular parts of Nav1.7 channel in regulation of its membrane expression.
Methods work plan:
We have fused the reporter protein GFP to the Nav1.7 channel. This allows us to use live imaging to track Nav1.7 localisation in sensory neurons. Nav1.7 on plasma membrane will be quantified in standard and inflammation-like conditions. Live imaging will be performed using the DeltaVision OMX super resolution system. Published reports and our preliminary research indicated that the N and C-termini of Nav1.7 may play a role in regulating its surface pool. DNA engineering will be used to produce N and C-termini mutants of the GFP-tagged channel to assess their contribution. Once the role of N and C-termini has been assessed, proteins that interact with them will be identified by a mass spectrometry. The role of the identified proteins will be validated by knockdown approaches. Lentiviruses will be used to introduce DNA coding for recombinant proteins and knockdown microRNA into sensory neurons. The project will involve using molecular biology methods to generate fusion protein constructs, transfection of DNA into cell lines and primary DRG neurons, immunocytochemistry and Western blotting. Functional effect of transfected fusions on Nav1.7 will be assessed by calcium imaging and patch clamping.
Impact:
The proposed work will provide a better understanding of Nav1.7 regulation in sensory neurons and may provide insights into pathways relevant to pathological changes in chronic pain conditions. Moreover, results may prove relevant to other membrane proteins in DRG whose surface pool could co-regulated with Nav1.7 by same pathways to decrease pain thresholds.