Regulation of K+-dependent Na+/Ca2+-exchanger subtype 4, NCKX4, by palmitoylation

• Published in: Cell calcium (Edinburgh) Vol. 131; p. 103069
• Main Authors: Al-Khannaq, By Maryam, Lytton, Jonathan
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.11.2025
• Subjects: Acyl-RAC; Ca2+ imaging; Ca2+ transport; Click chemistry; Colocalization; K+-dependent Na+/Ca2+-exchanger; Palmitoylation; Proximity ligation assay; Regulation; Colocalization; Palmitoylation; Ca2+ imaging; Proximity ligation assay; K+-dependent Na+/Ca2+-exchanger; Regulation; Acyl-RAC; Click chemistry; Ca2+ transport
• ISSN: 0143-4160
• DOI: 10.1016/j.ceca.2025.103069

•NCKX4 is palmitoylated on Cys118 and Cys425.•Palmitoylated NCKX4 has a subcellular distribution similar to total NCKX4 protein.•The level of NCKX4 palmitoylation can change more than two-fold within 6 h.•Altering the level of NCKX4 palmitoylation influences its subcellular distribution.•Altering palmitoylation has no effect on cellular NCKX4-mediated Ca2+ transport. Mammalian K+-dependent Na+/Ca2+ exchangers (NCKX), encoded by the SLC24 gene family, are crucial for maintaining Ca2+ homeostasis. NCKX4, widely expressed in the brain and sensory neurons, plays a key role in neuronal satiety and enamel formation. Despite its importance, the regulatory mechanisms of NCKX4 remain largely unexplored. This study investigates how palmitoylation, a post-translational modification affecting membrane proteins, regulates NCKX4 and influences its cellular localization and function. Using Acyl-RAC and palmitate-based click-chemistry, we found that approximately 14% of NCKX4 is palmitoylated at steady-state in both endogenous and transfected systems. The level of this modification is highly dynamic, being regulated by inhibitors of palmitoylation (2-bromopalmitate) and depalmitoylation (palmostatin B), resulting in greater than a two-fold decrease or increase, respectively. Site-directed mutagenesis of six cysteine residues revealed two key sites (Cys118 and Cys425) critical for NCKX4 palmitoylation. The subcellular distribution of palmitoylated NCKX4 was examined via proximity ligation and click-chemistry. NCKX4 was found across multiple membrane compartments, with a higher fraction localizing to the plasma membrane when palmitoylation was inhibited by 2-bromopalmitate. However, a Ca2+ imaging assay in HEK293T cells showed no significant change in aggregate cellular NCKX4-mediated Ca2+ transport upon modulation of palmitoylation status. These data suggest palmitoylation promotes internalization of the NCKX4 protein while also activating it, counter-acting effects that result in unchanged NCKX4-mediated cellular Ca2+ transport activity. In summary, NCKX4 is subject to dynamic palmitoylation, which influences both distribution across cellular compartments and intrinsic Ca2+ transport activity. These findings contribute to our understanding of the regulation and functional roles of NCKX4 in cellular physiology. [Display omitted]