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Cellular Dynamics of Voltage-gated Calcium Channel β Subunits
[Thesis]. Manchester, UK: The University of Manchester; 2012.
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Abstract
Calcium entry through voltage-gated calcium (CaV) channels is important in diverse cellular processes including neurotransmitter release, gene expression and cardiac pacemaker activity. CaV channels auxiliary CaVβ subunits enhance plasma membrane expression and modify the biophysical properties of CaVα1 subunits. Due to their multi-domain structures – including a conserved SH3-GK ‘core’ and hypervariable N- and C- terminal domains - CaVβs are also considered to be members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, and may therefore act as molecular scaffolds both within and outside the CaV channel complex. This project studied the roles of CaVβ N- and C-terminal hypervariable domains in contributing to isoform-specific differences in CaVβ functions both in a) CaV channel complex expression and distribution, and b) interactions with non channel proteins. To analyse such contributions a series of molecular tools were developed to assess the distributions of CaVβs (both within and outside the CaV channel complex) and their interactions with novel potential partner proteins. This involved systematically testing fluorophore- and epitope-tagged CaVβs for co localisation with both fluorophore-tagged CaV2.2 and a range of myc-tagged potential interaction partners (as quantified either by a ‘Membrane Localisation Index’ developed during this project or Intensity Correlation Analysis). This approach uncovered much detail about relative isoform specificities of CaVβ non-channel complex protein-protein interactions, however one particularly striking interaction was discovered between CaVβ1b/CaVβ4 and the nuclear protein Heterochromatin 1 γ (HP1γ), where nuclear translocation of CaVβ1b or CaVβ4 was induced upon association with HP1γ. Given the similarity of CaVβ1b and CaVβ4 N termini, a series of CaVβ1b N-terminal chimeras were then created, where the N terminus was exchanged with that of CaVβ3 (which did not interact with HP1γ). Subsequent imaging studies using these chimeras then confirmed that the CaVβ1b N terminus is necessary for co-localisation with HP1γ and subsequent HP1γ mediated CaVβ nuclear uptake.Given that an interaction between the CaVβ3 isoform and Pax6(S) - another nuclear protein - have been reported, where the CaVβ3–Pax6(S) interaction also induces nuclear translocation of both proteins, the CaVβ1b/CaVβ4-HP1γ interaction may represent one of a range of as-yet undiscovered CaVβ1b/gene regulatory protein interactions. As interaction with CaVβ3 suppresses the transcriptional activity of Pax6(S), nuclear targeting may be an important means by which CaVβs modulate gene expression – which in the case of HP1γ interactions may occur via de-repression.