S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:699923 |
| Date | January 2014 |
| Creators | Bi, Danlei |
| Contributors | Shipston, Michael ; Evans, Anthony |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17286 |
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