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Title: Trafficking of voltage-gated calcium channel subunits in sympathetic neurons
Author: Waithe, D. C.
ISNI:       0000 0004 2732 833X
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2009
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Voltage-gated calcium channels (VDCC) are essential for the correct functioning of all excitable cells. This project characterises the trafficking of N-type VDCC in response to two known modulators of channel expression and trafficking: Ca_\upsilon\beta subunits and \gamma7. Ca_\upsilon\beta subunits bind to an interaction site, the Alpha-Interaction Domain (AID), conserved in the I-II loop of the Ca_\upsilon\alpha1 subunit of all high-voltage-activated calcium channels. Mutation of W391 to A in the AID of Ca_\upsilon2.2, has identified that this amino acid is important for \beta subunit binding, for plasma membrane expression and for modulation of the biophysical properties of Ca_\upsilon2.2 channels. This study focuses on the localization of fluorescently tagged wild-type (WT) and W391A mutant Ca_\upsilon2.2 channels in sympathetic neurons. A significant difference in the localisation in neurites of Ca_\upsilon2.2(WT) compared to mutant channel Ca_\upsilon2.2(W391A) channels is shown, using co-expression techniques, photobleaching assays and time-series based live-cell imaging. The Ca_\upsilon2.2 channel distribution is sensitive to Ca_\upsilon\beta1b expression, although some of the distribution within the neurites occurs through a Ca_\upsilon\beta1b independent mechanism. The expression of \gamma7 has previously been shown to abolish N-type channel currents when co-expressed in either Xenopus oocytes or COS-7 cells. \gamma7 acts through a mechanism which modulates N-type channel expression by increasing the degradation rate of mRNA encoding the Ca_\upsilon2.2(WT) channel. The distribution of \gamma7 was investigated in sympathetic neurons to establish the localisation and trafficking of this protein. \gamma7 was found to be distributed throughout the neurites of the sympathetic neurons, as small puncta of fluorescence. In addition, \gamma7 expression was shown to initiate particle formation and trafficking, providing a possible link to the process of endocytosis or autophagy in neurites. This study has explored some of the mechanisms which regulate N-type VDCC trafficking and highlighted some of its key properties.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available