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

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).