Structures of a Constitutively Active Mutant of the Calcium-Activated Chloride Channel TMEM16A in Ligand-Free and -Bound States

Abstract

The binding of Ca2+ from the cytoplasm to the anion-selective channel TMEM16A triggers a conformational change around its binding site, which is coupled to the opening of an hourglass-shaped pore. We have previously identified three bulky hydrophobic residues at the intracellular entrance of the neck as constituents of the gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. Here, we determined cryo-electron microscopy structures of one of these constitutively active mutants in the absence and presence of Ca2+. Whereas the mutant resembles wild-type in the Ca2+-bound state, the Ca2+-free structure exhibits pronounced differences in the pore region compared to the corresponding structure of the wild-type channel. Similar to wild-type, the lower half of the Ca2+-binding and pore-lining helix α6 in the mutant adopts a ‘down’ conformation and directly coordinates the bound Ca2+ in the Ca2+-bound state. In the absence of Ca2+, this helix likewise dissociates from the binding site in the mutant, but the movement is less pronounced and proceeds in a different direction compared to wild-type. Consequently, α6 in the mutant remains in a position that is closer to its fully activated conformation, thereby lowering the energetic penalty for channel opening. Despite the difference in its position, the relaxation of α6 from a π- to an α-helix upon dissociation of Ca2+ is also observed in the mutant structures, suggesting that pore opening can proceed without a transition into the more strained π-helix conformation. Together, these observations emphasize a coupling between the gate and the Ca2+ binding element of α6, allowing Ca2+ binding to stabilize a conductive state of the channel, and provide a structural view of ligand-free activation of TMEM16A.