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Title: Trafficking of voltage-gated sodium channels involved in pain perception
Author: Hynes, Judith Audrey
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Chronic pain is a highly unmet clinical need; often treatment is only partially effective, and is frequently accompanied by unpleasant side effects. The voltage-gated sodium channel Nav1. 7 has emerged as an attractive target in the treatment of pain. Expressed preferentially in nociceptive DRG neurons, Nav1.7 functions at the plasma membrane to transmit peripherally generated pain signals towards the central nervous system. Manipulating Nav1.7 trafficking mechanisms to reduce the cell surface density of the channel is thus a promising approach to reduce the sensation of pain, yet there are no previous reports on Nay1.7 trafficking mechanisms. To allow investigation of human Nav1.7 trafficking mechanisms, epitope tags were inserted into the extracellular domains of the channel. Tagged channels expressed within the cells, but lacked surface expression when assayed by immunostaining. chemiluminescence assays and electrophysiology. Studies then focussed on the role of individual cytosolic domains of Nav1.7 - which harbour a number of consensus trafficking motifs- in the trafficking of the channel. Peptide domains of Nav1.7 were employed in a peptide competition assay to screen the domains for a dominant negative effect on the trafficking of the channel. Results from these assays were inconclusive. Internalisation of the full-length channel was demonstrated using a surface biotinylation assay. Chimeras of C04 and three of the cytosolic domains of Na,,1.7 were constructed to investigate the underlying mechanism of channel endocytosis. Using immunostaining assays, all three domains were shown to promote internalisation of C04, consistent with the presence of consensus endocytosis motifs in each. Furthermore, the Na,,1.7-C-terminal-CD4 chimera was shown to co-Iocalise with markers of early endosomes following endocytosis. These data provide the first evidence of endocytosis of Na.,.1.7 and have implicated a role for the cytosolic domains of the channel in regulating this process. Further elucidation of these mechanisms could reveal novel targets for the treatment of pain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available