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Updated: May 12, 2026
A Fluorescence-based Assay of Phospholipid Scramblase Activity
Published on: September 20, 2016
Calcium-dependent phospholipid scramblase activity of TMEM16 protein family members
Jun Suzuki1, Toshihiro Fujii, Takeshi Imao
1Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Background:
TMEM16A and 16B work as Cl(-) channel, whereas 16F works as phospholipid scramblase. The function of other TMEM16 members is unknown.
Results:
Using TMEM16F(-/-) cells, TMEM16C, 16D, 16F, 16G, and 16J were shown to be lipid scramblases.
Conclusion:
Some TMEM16 members are divided into two Cl(-) channels and five lipid scramblases.
Significance:
Learning the biochemical function ofTMEM16family members is essential to understand their physiological role. Asymmetrical distribution of phospholipids between the inner and outer plasma membrane leaflets is disrupted in various biological processes. We recently identified TMEM16F, an eight-transmembrane protein, as a Ca(2+)-dependent phospholipid scramblase that exposes phosphatidylserine (PS) to the cell surface. In this study, we established a mouse lymphocyte cell line with a floxed allele in the TMEM16F gene. When TMEM16F was deleted, these cells failed to expose PS in response to Ca(2+) ionophore, but PS exposure was elicited by Fas ligand treatment. We expressed other TMEM16 proteins in the TMEM16F(-/-) cells and found that not only TMEM16F, but also 16C, 16D, 16G, and 16J work as lipid scramblases with different preference to lipid substrates. On the other hand, a patch clamp analysis in 293T cells indicated that TMEM16A and 16B, but not other family members, acted as Ca(2+)-dependent Cl(-) channels. These results indicated that among 10 TMEM16 family members, 7 members could be divided into two subfamilies, Ca(2+)-dependent Cl(-) channels (16A and 16B) and Ca(2+)-dependent lipid scramblases (16C, 16D, 16F, 16G, and 16J).
Insights
The TMEM16 family has seven members: two chloride channels and five lipid scramblases. Understanding these TMEM16 proteins is key to their physiological roles.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The TMEM16 family comprises proteins with diverse functions, including ion channel activity and phospholipid scrambling.
- TMEM16A and TMEM16B are known chloride channels, while TMEM16F functions as a phospholipid scramblase.
- The specific roles of other TMEM16 family members remain largely uncharacterized.
Purpose of the Study:
- To elucidate the biochemical functions of TMEM16 family members.
- To classify TMEM16 proteins into distinct functional subfamilies based on their activity.
- To investigate the role of TMEM16F in phospholipid scrambling and its impact on phosphatidylserine exposure.
Main Methods:
- Generation of a TMEM16F knockout mouse lymphocyte cell line using a floxed allele system.
- Functional expression of various TMEM16 proteins in TMEM16F-deficient cells.
- Patch-clamp analysis to assess ion channel activity.
- Phosphatidylserine (PS) exposure assays using calcium ionophores and Fas ligand stimulation.
Main Results:
- TMEM16F-deficient cells failed to expose PS in response to calcium ionophores, confirming TMEM16F's role in PS exposure.
- Expression of TMEM16C, 16D, 16G, and 16J in TMEM16F(-/-) cells demonstrated their function as lipid scramblases with varying substrate preferences.
- Patch-clamp analysis identified TMEM16A and TMEM16B as distinct Ca(2+)-dependent chloride channels, while other family members did not exhibit this activity.
Conclusions:
- The TMEM16 family can be functionally divided into two subfamilies: Ca(2+)-dependent chloride channels (TMEM16A and TMEM16B) and Ca(2+)-dependent lipid scramblases (TMEM16C, 16D, 16F, 16G, and 16J).
- This classification provides a framework for understanding the diverse physiological roles of TMEM16 proteins.
- Elucidating the specific functions of each TMEM16 member is crucial for understanding their involvement in various biological processes.
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