BPS2026 - Using transition metal FRET to probe PIP2-mediated gating in TMEM16A

TransMEMbrane 16A (TMEM16A) is a broadly expressed chloride channel, regulated by both intracellular Ca 2+ and phosphatidylinositol 4,5-bisphosphate (PIP 2 ), an acidic phospholipid found in the inner leaflet of eukaryotic cells. The channel is a homodimer, with each subunit comprising ten transmembrane spanning domains (TMs) and a membrane-embedded Ca 2+ binding site located within TMs 6–8. Although multiple TMEM16A structures have been published, they capture the channel in a closed conformation without resolved PIP 2 , leaving the structural basis of PIP 2 regulation unknown. To address this, we used transition metal Förster resonance energy transfer (tmFRET) as a molecular ruler to probe PIP 2 -dependent conformational changes. Ni 2+ binds and activates TMEM16A at the Ca 2+ binding site and serves as the FRET acceptor, while the fluorescent unnatural amino acid, Anap, functions as the donor. Because tmFRET is sensitive to distances of 10–30 Å, this approach provides a sensitive measure of the distance between Anap-labeled residues and the Ni 2+ -occupied binding site, enabling us to monitor structural rearrangements in TMEM16A upon manipulation of PIP 2 levels. Anap was engineered into multiple sites within TMs 3–6 in the full-length mouse isoform of TMEM16A channels and expressed in HEK293T cells. In control experiments, we observed higher tmFRET efficiency ( N = 6) when Anap was inserted near the Ca 2+ binding site, F653. By contrast, Anap inserted far from the Ca 2+ binding site, at position Y458 (TMs 2–3 intracellular linker), exhibited little tmFRET. We found that the tmFRET efficiency of Anap inserted in TM6 (F653) decreased with PIP 2 depletion by neomycin, suggesting that PIP 2 induces movement of TM6 toward the Ca 2+ binding site. These results provide evidence of PIP 2 -induced conformational changes in TMEM16A and establish a framework for elucidating the molecular mechanisms governing lipid regulation of this channel.