Distinct structural mechanisms drive gain-of-function activation of TMEM16E in gnathodiaphyseal dysplasia

In cells TMEM16E (ANO5) mediates Ca²⁺-dependent currents and lipid scrambling. Its mutations cause muscular dystrophies and gnathodiaphyseal dysplasia 1 (GDD), but its activation mechanism is unknown. We show purified TMEM16E is a scramblase and determined cryoEM structures in apo and Ca²⁺-bound states. Unlike other TMEM16s, apo TMEM16E has a straight TM6 helix and preformed orthosteric sites; Ca²⁺ binding induces no rearrangements, and one site remains partially unoccupied in saturating Ca²⁺. Structures of two GDD gain-of-function mutants, G503E and R582I, show that despite similar functional phenotypes they act through distinct mechanisms: G503E disrupts the TM3-TM4 interface, while R582I remodels an extracellular loop network and increases S2 occupancy. Molecular dynamics simulations show G503E favors an X-shaped groove that scrambles lipids outside the groove, resembling active TMEM16F, rather than the open-groove mechanism of the ER-resident TMEM16K. Thus, phenotypically convergent mutations act through distinct pathways, paving the way for development of targeted therapies. .