Meyer, James Frederick Otto;
(2019)
Exploring the mechanisms that regulate CaV2.2 trafficking and function.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
N-type calcium channels (CaV2.2) are pseudotetrameric voltage-gated calcium channels (VGCCs) predominantly expressed at the presynaptic terminals in the peripheral and central nervous system. CaV2.2 channels are highly calcium selective and play a crucial role in neurotransmitter release, coupling extracellular calcium entry to neurotransmitter exocytosis. The activity of CaV2.2 has multiple forms of regulation including: post-translational modifications (PMTs), proteolytic degradation and subcellular localisation. In addition, the behaviour of VGCCs is modulated by their auxiliary α2δ and β subunits. In this study, I first examine the role of proteolytic α2δ processing on CaV2.2. I find that unprocessed α2δ subunits, while unable to enhance whole-cell CaV2.2 currents, retain the ability to promote cell-surface expression of CaV2.2 in cell lines. Subsequent restoration of α2δ processing did not influence the protein expression of α2δ or of coexpressed CaV2.2. Thereafter, I examined the properties of CaV2.2 mutants featuring substitutions of crucial P-loop glutamate residues within the selectivity filter. I find that mutation of these glutamate residues renders these channels deficient in trafficking both in non-neuronal cell lines and primary neuronal cultures. These data are pertinent given the use of P-loop CaV mutants as dominant negative channels in previous studies. Finally, I compare the influence of α2δ-1, α2δ-2 and α2δ-3 subtypes on the cell-surface and total expression of CaV2.2. Both cell-surface and total expression of CaV2.2 was found to be enhanced by all three subtypes albeit to differing degrees. Further investigation reveals that α2δ-1 and α2δ-2 promote the net forward trafficking of CaV2.2 through Rab11a-dependent recycling. CaV2.2 does not appear to participate in Rab11a-dependent recycling when expressed in the absence of α2δ or with α2δ-3. This study reveals differential regulation of CaV2.2 channels among α2δ subtypes through targeted endosomal trafficking.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Exploring the mechanisms that regulate CaV2.2 trafficking and function |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/ 4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10075882 |
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