Regulation of K+ -dependent Na+/Ca2+ -exchanger subtype 4, NCKX4, by palmitoylation
Date
2024-06-28
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Abstract
Mammalian K+-dependent Na+/Ca2+-exchangers (NCKX), encoded by the SLC24 gene family, are Ca2+ extrusion proteins crucial for Ca2+ homeostasis. NCKX4 is widely expressed, particularly in the brain, and is significant in visual and olfactory sensory neurons, neuronal satiety pathways, and dental enamel formation. The regulation of NCKX4 remains largely unexplored. Investigating dynamic regulation of NCKX4 will further our understanding of Ca2+ homeostasis in biological functions and may identify therapeutic targets for eating disorders. Palmitoylation is a post-translational modification that regulates many features of membrane proteins. This study investigates NCKX4 regulation by palmitoylation and examines its effects on NCKX4 cellular localization and function. Understanding NCXK4 palmitoylation is of fundamental importance for advancing our knowledge of transporter regulation and function. Using Acyl-RAC and palmitate-based click-chemistry methods, it was found that approximately 14% of NCKX4 is palmitoylated in both endogenous and transfected systems. This level of palmitoylation was dynamically regulated and could be decreased and increased by inhibitors of palmitoylation (2-bromopalmitate; 2BP) and depalmitoylation (palmostatin B; palmB), respectively. Palmitoylation sites were determined using site-directed mutagenesis of six cysteines, two of which (C118S and C425S) significantly reduced the level of NCKX4 palmitoylation. However, the C118S/C425S double mutant did not eliminate all palmitoylation, indicating the presence of additional palmitoylation sites in NCKX4. The cellular localization of palmitoylated NCKX4 was examined using palmitate-based click-chemistry combined with proximity ligation. Palmitoylated NCKX4 is distributed across multiple cellular membrane compartments. The effect of palmitoylation on NCKX4 cellular localization was examined using 2BP and PalmB with Na+/K+ -ATPase as a plasma membrane marker. The data show a significantly higher fraction of NCKX4 is found in the plasma membrane when palmitoylation is inhibited with 2BP. The effect of palmitoylation on NCKX4 activity was investigated using a Ca2+ imaging assay in HEK293T and MEB4 cells treated with 2BP and PalmB. No significant difference was found in the activity NCKX4 under these different conditions. In conclusion, NCKX4 is palmitoylated at several sites on the protein. The extent of palmitoylation is dynamically regulated, and palmitoylation affects NCKX4 cellular distribution, while it has no measurable effect on aggregate NCKX4 Ca2+ transport activity.
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Keywords
Calcium, ion exchangers, NCKX, protein regulation, palmitoylation, lipidation
Citation
Al-Khannaq, M. (2024). Regulation of K+ -dependent Na+/Ca2+ -exchanger subtype 4, NCKX4, by palmitoylation (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.