Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA050

Poster Communications

The Impact of Palmitoylation on Na+-Ca2+ Exchanger (NCX) Function

C. Gok1, W. Fuller1

1. Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom.

The sodium-calcium (Na+-Ca2+) exchanger (NCX) is an antiporter membrane protein that regulates cytoplasmic Ca2+ by facilitating its electrogenic exchange for 3 Na+. Cardiac NCX (NCX1) is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. A single cysteine in position 739 of the NCX1 large intracellular loop is palmitoylated, which regulates NCX1 inactivation (Reilly L et al. (2015). FASEB J. 4532-4543). We investigated the impact of palmitoylation on NCX1 function using resin-assisted capture, and Fluorescence Resonance Energy Transfer (FRET) in HEK293 cells transfected with wild type (WT) or unpalmitoylatable (C739A) NCX1. NCX1 functions as a dimer: interactions between NCX1 dimers were visualised as FRET changes between exchanger proteins with CFP or YFP inserted at position 266 within the large cytoplasmic loop. NCX1-NCX1 FRET was greater in cells expressing WT than C739A NCX1, and the broad-spectrum palmitoylation inhibitor 2-Bromopalmitate reduced NCX1-NCX1 FRET only in cells expressing WT NCX1. Affinity purification followed by mass spectrometry identified the thioesterase APT1 as an NCX1-interacting protein: APT1 inhibition increased NCX1 palmitoylation and NCX1-NCX1 FRET. The palmitoylation of NCX1 and NCX1-NCX1 FRET were altered by the ion composition of the extracellular buffer: Ca-free extracellular solutions favoured FRET and elevated NCX1 palmitoylation, whereas Na-free extracellular solutions did the opposite. C739A-NCX1 FRET was unaffected by Ca-free conditions. We conclude that palmitoylation influences the structure of the NCX1 intracellular loop to modify FRET between NCX1 dimers. The impact of extracellular solution composition on NCX1 palmitoylation implies the existence of mechanisms to dynamically couple NCX1 palmitoylation to its function.

Where applicable, experiments conform with Society ethical requirements