Cells establish the asymmetrical distribution of phospholipids and alter their distribution by phospholipid scrambling (PLS) to adapt to environmental changes. Here, we demonstrate that a protein complex, consisting of the ion channel Tmem63b and the thiamine transporter Slc19a2, induces PLS upon calcium (Ca²⁺) stimulation. Through revival screening using a CRISPR sgRNA library on high PLS cells, we identify Tmem63b as a PLS-inducing factor. Ca²⁺ stimulation-mediated PLS is suppressed by deletion of Tmem63b, while human disease-related Tmem63b mutants induce constitutive PLS. To search for a molecular link between Ca²⁺ stimulation and PLS, we perform revival screening on Tmem63b-overexpressing cells, and identify Slc19a2 and the Ca²⁺-activated K⁺ channel Kcnn4 as PLS-regulating factors. Deletion of either of these genes decreases PLS activity. Biochemical screening indicates that Tmem63b and Slc19a2 form a heterodimer. These results demonstrate that a Tmem63b/Slc19a2 heterodimer induces PLS upon Ca²⁺ stimulation, along with Kcnn4 activation.

J. Suzuki, H. Niu, and M. Maruoka are inventors on a patent application of Ca²⁺-dependent phospholipid scrambling. The remaining authors declare no competing interests.