Functional Swapping between Transmembrane Protein TMEM16A and TMEM16F [Membrane Biology]

January 29th, 2014 by Suzuki, T., Suzuki, J., Nagata, S.

The transmembrane proteins TMEM16A and 16F each carry eight transmembrane regions with cytoplasmic N- and C-termini. TMEM16A carries out Ca2+-dependent Cl- ion transport, and TMEM16F is responsible for Ca2+-dependent phospholipid scrambling. We here established an assay system for the Ca2+-dependent Cl- channel activity using 293T cells, and for the phospholipid scramblase activity using TMEM16F-/- immortalized fetal thymocytes (IFETs). Chemical cross-linking analysis showed that TMEM16A and 16F form homodimers in both 293T cells and IFETs. Successive deletion from the N- or C-terminus of both proteins and the swapping of regions between TMEM16A and 16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F), and C- terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and 16F mutants with point mutations in the pore region (located between the 5th and 6th transmembrane regions) indicated that the pore region is essential for both 16A′s Cl- channel activity and 16F′s phospholipid scramblase activity. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited TMEM16A′s Cl- channel activity and 16F′s scramblase activity with an opposite preference. These results indicate that TMEM16A and 16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains may significantly differ.