An inner activation gate controls TMEM16F phospholipid scrambling

Area of Science:

• Biochemistry
• Molecular Biology
• Membrane Protein Function

Background:

• Transmembrane protein 16F (TMEM16F) functions as a calcium-activated phospholipid scramblase (CaPLSase), crucial for processes like blood coagulation and bone development.
• Understanding the gating mechanism of TMEM16F, particularly how its phospholipid permeation pathway opens and closes, is essential but remains largely unclear.

Purpose of the Study:

• To identify and characterize the gating mechanism of the TMEM16F phospholipid scramblase.
• To elucidate the structural basis for calcium-dependent activation and phospholipid permeation in TMEM16F.

Main Methods:

• Site-directed mutagenesis was used to investigate the role of specific residues in TMEM16F function.
• Functional assays were performed to assess phospholipid scrambling activity and calcium-dependent activation.

Main Results:

• An inner activation gate, comprising hydrophobic residues F518, Y563, and I612, was identified within the TMEM16F permeation pathway.
• Disruption of this inner gate significantly altered TMEM16F phospholipid permeation.
• Specific lysine substitutions (F518K, Y563K) resulted in constitutively active TMEM16F, bypassing calcium-dependent activation.
• A homologous mutation in TMEM16A (L543K) conferred CaPLSase activity, suggesting conserved gating principles.

Conclusions:

• The identified inner activation gate is critical for regulating TMEM16F phospholipid scramblase activity.
• Understanding this gate provides mechanistic insights into the gating and permeation of TMEM16 family proteins.
• This discovery opens avenues for exploring TMEM16A channel function and TMEM16F scramblase regulation.