Phosphatidylserine (PS) is normally sequestered in the inner leaflet of the plasma membrane and its surface exposure triggers blood clotting by activated platelets and marks apoptotic cells for phagocytic clearance. PS exposure is mediated in part by Ca2+-dependent lipid scramblases that flip lipids across the plasma membrane. Despite their importance in cell physiology, the molecular identity of the scramblases has eluded researchers for decades. Recently, TMEM16F, a member of the TMEM16 family of Ca2+-activated Cl- channels, was shown to be important for Ca2+-dependent PS exposure. Mutations in TMEM16F cause Scott syndrome, an inherited bleeding disorder associated with defective lipid scrambling in platelets. TMEM16F-null mice recapitulate this disorder, in addition to displaying other defects, such as decreased bone mineralization. Although TMEM16F is important for phospholipid scrambling in platelets, its role in the process remains unclear and controversial: it has been claimed to be a scramblase, an ion channel, or a dual function protein with both scramblase and channel activity. Our long-term goal is to elucidate the molecular bases of lipid scrambling by TMEM16F and its regulation by Ca2+. These insights will allow us to understand the etiology of Scott syndrome and the role of TMEM16F in this disease as well as in other processes. To achieve our overall goal we propose to identify the structural basis for ion and lipid transport in TMEM16 proteins, determine the function and physiological role of TMEM16F and elucidate the molecular basis of Ca2+ sensing in TMEM16 proteins. Our approach is to combine biochemical assays on purified proteins with lipid scrambling and electrophysiological measurements of the same proteins in cells. We recently succeeded in expressing, purifying and functionally reconstituting TMEM16 proteins to demonstrate their intrinsic scramblase and channel activities. We also showed that expression of TMEM16F in cells indeed leads to ion transport and lipid scrambling. Thus, we have a strong platform of preliminary data to support our approach. Our proposal to understand lipid scrambling and the physiological functions of TMEM16 proteins is highly significant, as it will identify the molecular basis of Scott syndrome and elucidate the fundamental mechanism of regulated transbilayer lipid transport, a process that is not understood in any system.

Public Health Relevance

Scramblase proteins expose phosphatidylserine on the surface of cells, enabling activated platelets to trigger blood coagulation and phagocytes to eliminate cells undergoing programmed cell death. TMEM16 proteins are important for scrambling and their deficiency is associated with the bleeding disorder known as Scott syndrome. Our goal is to understand how the TMEM16 proteins work and to elucidate the etiology of Scott syndrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM106717-01A1
Application #
8728513
Study Section
Special Emphasis Panel (ZRG1-CB-G (02))
Program Officer
Chin, Jean
Project Start
2014-06-04
Project End
2018-02-28
Budget Start
2014-06-04
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
$574,236
Indirect Cost
$215,212
Name
Weill Medical College of Cornell University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065