Projects per year
Abstract
The electrogenic sodium/calcium exchanger (NCX) mediates bidirectional calcium transport controlled by the transmembrane sodium gradient. NCX inactivation occurs in the absence of phosphatidylinositol 4,5-bisphosphate (PIP2), and is facilitated by palmitoylation of a single cysteine at position 739 within NCX's large intracellular loop. The aim of this investigation was to identify the structural determinants of NCX1 palmitoylation. Full-length NCX1 (FL-NCX1) and a YFP fusion protein of the NCX1 large intracellular loop (YFP-NCX1) were expressed in HEK cells. Single amino acid changes around C739 in FL-NCX1 and deletions on the N-terminal side of C739 in YFP-NCX1 did not affect NCX1 palmitoylation, with the exception of the rare human polymorphism S738F, which enhanced FL-NCX1 palmitoylation, and D741A, which modestly reduced it. In contrast, deletion of a 21-amino-acid segment enriched in aromatic amino acids on the C-terminal side of C739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate C739 palmitoylation. Introduction of negatively charged amino acids to the hydrophobic face or of helix-breaking prolines impaired palmitoylation of both YFP-NCX1 and FL-NCX1. Alanine mutations on the hydrophilic face of the helix significantly reduced FL-NCX1 palmitoylation. Of note, when the helix-containing segment was introduced adjacent to cysteines that are not normally palmitoylated, they became palmitoylation sites. In conclusion, we have identified an amphipathic α-helix in the NCX1 large intracellular loop that controls NCX1 palmitoylation. NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence.
Original language | English |
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Pages (from-to) | 10745-10752 |
Number of pages | 8 |
Journal | Journal of Biological Chemistry |
Volume | 292 |
Issue number | 25 |
Early online date | 21 Apr 2017 |
DOIs | |
Publication status | Published - 23 Jun 2017 |
Keywords
- sodium-calcium exchange
- Protein acylation
- Protein palmitoylation
- Calcium transport
- Sodium transport
- Acyltransferase
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Dive into the research topics of 'An Amphipathic α-Helix Directs Palmitoylation of the Large Intracellular Loop of the Sodium/Calcium Exchanger'. Together they form a unique fingerprint.Projects
- 4 Finished
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Palmitoylation of the L-Type Ca Channel Pore-Forming Subunit (joint with University of Bristol)
Fuller, W. (Investigator) & Hales, T. (Investigator)
29/08/16 → 27/02/18
Project: Research
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Characterisation of the Cardiac Palmitoyl Transferase DHHC5 (PhD Studentship)
Fraser, N. (Investigator) & Fuller, W. (Investigator)
13/07/15 → 12/07/18
Project: Research
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Interaction Between Palmitoylation and Glutathionylation in the Regulation of Cardiac Function
Fuller, W. (Investigator) & Henderson, C. (Investigator)
1/08/13 → 31/07/16
Project: Research