Palmitoylation and regulation of potassium-dependent sodium/calcium exchangers (NCKX)

Abstract

Cellular Ca2+ homeostasis is critical for normal cell physiology and is regulated by several mechanisms. Two major players in intracellular Ca2+ homeostasis in multiple tissues belong to the SLC8 (Na+/Ca2+ exchangers (NCXs); NCX1-3) and SLC24 (K+ dependent Na+/Ca2+ exchangers (NCKXs); NCKX1-5) families. It has been established that NCXs and NCKX4 are palmitoylated, and that palmitoylation promotes NCX1 inactivation. However, there is still little known about NCKXs' palmitoylation. We found that (1) NCKX3 and NCKX5, but not NCKX1, are palmitoylated, (2) Cys to Ala mutation at position 467 for NCXK3 and 325 for NCKX5 notably diminished palmitoylation and (3) reduced palmitoylation enhanced NCKX3 activity. Our findings bring novel insights into NCKX1, NCKX3 and NCKX5 palmitoylation and establish palmitoylation as an endogenous regulator of NCKX3 activity, paving the way for investigations evaluating the role of palmitoylation in NCKX3 function in health and disease.

Keywords: Ca2+homeostasis; NCKX; Solute Carrier (SLC) proteins; palmitoylation.

Figure 1:. Understanding NCKXs’ palmitoylation.

(A) NCKX3 and NCKX5, unlike NCKX1, are palmitoylated in HEK293 cells transiently transfected with FLAG-tagged NCKXs (red arrowhead: NCKX1, green arrowhead: NCKX3, blue arrowhead: NCKX5, N:8). (B) AlphaFold prediction of NCKX3 (AF-Q9HC58) and NCKX5 (AF-Q71RS6) structure suggested potential (palmitoylatable) Cys residue adjacent to the α-helix (C467 for NCKX3 and C325 for NCKX5) and close to the membrane. Protein structures were built in ChimeraX [24] (C) Resin -assisted capture of palmitoylated NCKX3 and NCKX5 from HEK293 cells transiently transfected FLAG-tagged NCKX3 and NCKX5 showed that mutating C467 for NCKX3 and C325 for NCKX5 to Ala significantly reduced palmitoylation (N:5 for NCKX3 and N:4 for NCKX5, unpaired t-test; P<0.0001 for WT- vs. C467A- NCKX3 and P=0.0659 for WT- vs. C325A- NCKX5). UF: Uunfractionated cell lysate, HA: palmitoylated proteins, NaCl: Nnegative control.

Figure 2:. NCKX1 remains unpalmitoylated following V928C mutation.

(A) Clustal alignment (Clustal Ω) [26]) of NCKX1, NCKX3 and NCKX5 identified Val (underlined in pink) at NCKX1 position 928 in an analogous position to Cys residues (underlined in red) situated adjacent to the α-helix in both NCKX3 and NCKX5 structures. (B) Predicted NCKX1 structure (AF-O60721) built in ChimeraX [24]. (C) V928C mutation did not induce NCKX1 palmitoylation (red arrowheads, N:4). UF: Uunfractionated cell lysate, HA: palmitoylated proteins, NaCl: Nnegative control.

Figure 3:. Effect of palmitoylation on subcellular localization.

(A) Cell surface abundance of WT- and C467A- NCKX3 was similar (unpaired t-test; P=0.322 for WT- vs. C467A- NCKX3, N:4). (B) WT- (left) and C467A- NCKX3 (right) exhibited similar subcellular localization pattern in Tet- inducible engineered cells: little clusters scattered in the cytoplasm and nucleus (red: FLAG- NCKX3, and blue: Hoechst- nucleus). (C) NCKX5 is not present in the plasma membrane. The Na pump is a surface membrane protein and was used to confirm successful purification of surface membrane proteins. Streptavidin- HRP was used to confirm ifwhether surface proteins were successfully probed with biotin and pulled down/captured by streptavidin beads. UF: Uunfractionated cell lysate, UB: unbound fraction, B: Ppurified surface proteins.

Figure 4:. Disrupting NCKX3 palmitoylation increased NCKX3-mediated Ca²⁺ influx.

(A) Diagram illustrating the NCKX3 activity assay,. (B, C) NCKX3 -mediated Ca²⁺ influx was greater in C467A- NCKX3 compared towith WT in Tet- inducible engineered cells upon switching from Ca²⁺-free buffer to 1 mM or 5 mM Ca²⁺-containing buffer (1 mM Ca²⁺: one-way ANOVA with Tukey’s multiple comparison test; P=0.0022 for WT (+TET) - vs. C467A (+TET)- NCKX3; 5 mM: one-way ANOVA with Tukey’s multiple comparison test; P=0.0031 for WT (+TET) - vs. C467A (+TET)- NCKX3, N:4 and n:32 for each). Ca²⁺ intake was represented/calculated as the area under curve (AUC).