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

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.