Elsevier

Cellular Signalling

Volume 76, December 2020, 109791
Cellular Signalling

Review
Topical review: Shedding light on molecular and cellular consequences of NCX1 palmitoylation

https://doi.org/10.1016/j.cellsig.2020.109791Get rights and content

Highlights

  • NCX1 palmitoylation is directed by an amphipathic α-helix in its regulatory loop.

  • Palmitoylation modifies the NCX1 dimer's structure and affinity for lipid rafts.

  • NCX1 is dynamically palmitoylated at the cell surface by zDHHC5 and APT1.

  • Palmitoylation controls the affinity of the XIP domain for NCX1's regulatory loop.

  • By regulating XIP affinity palmitoylation regulates NCX1 inactivation & cellular Ca.

Abstract

Palmitoylation is the post-translational, covalent and reversible conjugation of a 16C saturated fatty acid to cysteine residues of proteins. The sodium calcium exchanger NCX1 is palmitoylated at a single cysteine residue in its large regulatory intracellular loop. Inactivation, mediated by the NCX1 inhibitory region XIP, is drastically impaired in unpalmitoylatable NCX1. The ability of XIP to bind and inactivate NCX1 is largely determined by NCX1 palmitoylation, which induces local conformational changes in the NCX1 intracellular loop to enable XIP to engage its binding site. Consequently, NCX1 palmitoylation regulates intracellular calcium by changing NCX1 sensitivity to inactivation. NCX1 palmitoylation is a dynamic phenomenon which is catalyzed by the palmitoyl acyl transferase zDHHC5 and reversed by the thioesterase APT1, with the switch between palmitoylated and depalmitoylated states, which has profound effects on NCX1 lipid interactions, influenced by NCX1 conformational poise. Herein we review the molecular and cellular consequences of NCX1 palmitoylation and its physiological relevance and highlight the importance of palmitoylation for NCX1 activity. We discuss the cellular control of protein palmitoylation and depalmitoylation, the relationship between lipid microdomains and lipidated and phospholipid binding proteins, and highlight the important unanswered questions in this emerging field.

Introduction

Developing interest in palmitoylation and its physiological consequences in different tissues over the last decade has enabled palmitoylation to gain increasing popularity and put a spotlight on this unique post-translational modification. Simply put, palmitoylation is enzymatically controlled phenomenon involving the dynamic attachment of palmitic acid to a target protein via a thioester bond. This unique biochemical modification in protein structure to date, has been reported with a regulatory role in several cellular and physiological processes in various tissues [[1], [2], [3]]. Despite the rich background regarding the role of palmitoylation in the nervous system [3], how palmitoylation contributes to cellular events in the cardiac tissue; such as calcium homeostasis and cardiac contractility, is poorly understood.

Various ion channels/transporters and signalling molecules; which work in harmony as a team, lie at “the heart” of cardiac tissue. How this complex team functions to maintain cellular calcium fluxes in balance to support healthy cardiac function is vital question to be addressed. Among hundreds of proteins in the cardiac system, cardiac Na+/Ca2+ Exchanger (NCX1) is established as a major player in calcium homeostasis and cardiac contractility. NCX1 mediates ~30% of cytosolic calcium removal in ventricles in large mammals [4]. NCX1 also acts as a part of rhythmic generator of action potential, the calcium clock, in the sinoatrial node (SAN) [5]. Inappropriate NCX1 behaviour is implicated in various cardiac pathologies with severe consequences [[6], [7], [8], [9]].

NCX1 is 10 transmembrane (TM) domain protein with a large intracellular loop between TM5 and TM6. This intracellular loop is a key regulator of NCX1 function and is composed of Exchanger Inhibitory Peptide (XIP), Calcium Binding Domains (CBDs; CBD1 and CBD2) and the palmitoylation site (Fig. 1) [[10], [11], [12], [13], [14], [15], [16]]. These regions promote different physiological states of the exchanger; XIP inhibits while calcium binding to CBDs activates the transporter [10,11,[17], [18], [19]]. Apart from the allosteric regulation of activation-inactivation states of NCX1 and the auto-regulation by XIP domain, palmitoylation is the first post-translational modification to be identified as a direct regulatory mechanism in NCX1 physiology [[13], [14], [15], [16],20]. Although the discovery of NCX1 palmitoylation and its role in the exchanger function are relatively new to the field, the initial findings are promising for palmitoylation to be a powerful tool to tune NCX1 physiology. Herein we summarize physiological, molecular and cellular consequences of NCX1 palmitoylation updated with recent findings.

Section snippets

Brief look up: what is currently known about NCX1 palmitoylation and its physiological consequences?

NCX1 possesses multiple cysteine residues in its structure but exclusively only one palmitoylatable cysteine was detected at position 739 in the large intracellular loop of the exchanger (Fig. 1) [13]. Abolishing NCX1 palmitoylation by introducing a single mutation at this site from cysteine to alanine (C739A) causes NCX1 to be unable to inactivate properly. Two separate experimental approaches using patch clamp in the whole cell configuration showed that a lack of palmitoylation disrupts the

Palmitoylation induces conformational changes in the intracellular loop of NCX1

The existence of an NCX1 dimer in plasma membrane is well-established [21]. The functional importance of NCX1 dimerisation is currently uncertain, but restructuring of the dimer appears to have important consequences for NCX1 regulation. Calcium dependent structural re-arrangements within the large intracellular loop of the exchanger were probed in oocyte membrane sheets using FRET sensors generated by insertion of either YFP or CFP to full length NCX1 at position 266, immediately after the XIP

Conclusion & future perspectives

Recent findings highlighted here have brought a new dimension into the understanding of the role for palmitoylation in NCX1 function (Fig. 3). NCX1 is dynamically palmitoylated at the cell surface by zDHHC5 and depalmitoylated by APT1. Palmitoylation is a key regulatory process for NCX1 inactivation and the control of intracellular calcium as well as for NCX1 interaction with lipid domains. However, the cellular events that control NCX1 palmitoylation and depalmitoylation as well as some

Declaration of Competing Interest

None.

Acknowledgements

We acknowledge financial support from the British Heart Foundation: SP/16/3/32317 to WF.

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