Interaction of human phospholipid scramblase 1 with cholesterol via CRAC motif is essential for functional regulation and subcellular localization

Human phospholipid scramblase 1 (hPLSCR1) possesses a putative cholesterol binding CRAC (cholesterol interaction/recognition amino acid consensus) motif at the C-terminal. The CRAC motif of hPLSCR1 interacts with cholesterol with an energy of interaction −64.39 KJ mol−1. Since palmitoylated hPLSCR1 localizes to the cholesterol-rich lipid rafts, the interaction between hPLSCR1 and raft cholesterol is highly likely. The present study investigated the hPLSCR1-cholesterol interaction in plasma membrane via putative CRAC motif. hPLSCR1 remains at cholesterol-rich lipid rafts as long as they interact. This interaction is inhibited by mutations in the CRAC motif or cholesterol depletion. Thus, CRAC mutants I300D hPLSCR1 and ΔCRAC hPLSCR1 diffused to the cytoplasm and nucleus. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) dose-dependently reduced cell viability in A549 cells. However, cholesterol depletion released 1.74 ± 0.12 times Ca2+ to the cytosol in A549 cells. Similarly, cholesterol depletion increased intracellular Ca2+ release by 1.81 ± 0.13 and 4.11 ± 0.19 times in RAJI cells expressing hPLSCR1 and ΔCRAC hPLSCR1, respectively. Moreover, the expression of hPLSCR1 and ΔCRAC hPLSCR1 increased apoptosis in RAJI cells by 21 ± 1.5% and 53.50 ± 4.40%, respectively. It was further increased to 43 ± 2.5% and 71.4 ± 1.4% upon cholesterol depletion. The current work links hPLSCR1 expression with cholesterol depletion, intracellular Ca2+ release, and induction of apoptosis. •Inhibition of hPLSCR1-cholesterol interaction import hPLSCR1 to the nucleus.•Mutation in the CRAC motif inhibits hPLSCR1-cholesterol interaction.•Depletion of membrane cholesterol inhibits hPLSCR1-cholesterol interaction.•hPLSCR1 induces IP3R1 expression inside the nucleus.•hPLSCR1 induces apoptosis in RAJI cells.