Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse
physiological processes. The nhTMEM16 scramblase (from the fungus Nectria haematococca) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides
the transbilayer conduit for lipids, but the mechanism by which Ca2+ regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity,
we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca2+-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the
nhTMEM16 homodimer, and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing
an acyl carrier protein (ACP)-tag at one or both of termini. These constructs were fluorescently labeled by ACP synthase-mediated
insertion of Coenzyme A-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the
distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor
analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer.
We now report that the measured distances do not vary significantly between Ca2+-replete and EGTA-treated samples, indicating that while the cytoplasmic portion of the protein is important for function,
it does not appear to regulate scramblase activity via a detectable conformational change.