An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger

Plain, F., Congreve, S. D., Yee, R. S. Z., Kennedy, J., Howie, J., Kuo, C.-W., Fraser, N. J. and Fuller, W. (2017) An amphipathic α-helix directs palmitoylation of the large intracellular loop of the sodium/calcium exchanger. Journal of Biological Chemistry, 292(25), pp. 10745-10752. (doi: 10.1074/jbc.M116.773945) (PMID:28432123) (PMCID:PMC5481580)

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 and is facilitated by palmitoylation of a single cysteine at position 739 within the large intracellular loop of NCX. 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 Cys-739 in FL-NCX1 and deletions on the N-terminal side of Cys-739 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 Cys-739 abolished YFP-NCX1 palmitoylation. We hypothesized that this segment forms an amphipathic α-helix whose properties facilitate Cys-739 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.

• Item Type: Articles
• Additional Information: This work was supported by British Heart Foundation Grants RG/12/4/29426 and PG/16/33/32134 (to W. F.) and Grant FS/14/68/30988 (to W. F. and N. J. F.)
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Fuller, Professor Will
• Authors: Plain, F., Congreve, S. D., Yee, R. S. Z., Kennedy, J., Howie, J., Kuo, C.-W., Fraser, N. J., and Fuller, W.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Journal of Biological Chemistry
• Publisher: American Society for Biochemistry and Molecular Biology, Inc.
• ISSN: 0021-9258
• ISSN (Online): 1083-351X
• Published Online: 21 April 2017
• Copyright Holders: Copyright © 2017 The American Society for Biochemistry and Molecular Biology, Inc.
• First Published: First published in Journal of Biological Chemistry 292(25):10745-10752
• Publisher Policy: Reproduced under a Creative Commons License