Final Report Summary - ANOBEST (Structure function and pharmacology of calcium-activated chloride channels: Anoctamins and Bestrophins)
Membranes form the borders enclosing cells and intracellular compartments. They are composed of lipids that assemble into bilayers, which restrict the flow of ions and polar substances. Despite these barriers, cells have to communicate with their environment and exchange substances that are essential for the survival of the organism via protein-mediated membrane transport processes. Two seemingly unrelated transport processes play a key role in important physiological functions. One concerns ion conduction through Ca2+-activated Cl- channels. These proteins open an anion-selective pore in response to the raise of intracellular Ca2+, thereby contributing to the flow of anions across epithelia and the control of electrical excitability. The other process concerns the exchange of lipids between the two leaflets of a bilayer catalyzed by lipid-scramblases. Lipid scrambling dissipates the asymmetry of the membrane to initiate important processes such as blood coagulation, cell-fusion and apoptosis. Although both processes have been investigated for decades, the involved proteins remained for a long time elusive. In the course of the ERC grant AnoBest, we have characterized the detailed mechanistic relationship between the two processes, which are both catalyzed by the TMEM16 family of membrane proteins. The family includes such important members as the chloride channel TMEM16A, whose expression in airway epithelia makes it an important pharmaceutical target for the treatment of cystic fibrosis, and the protein TMEM16F, which is a major player in the initiation of blood coagulation and whose malfunction underlies severe bleeding disorders. Our studies have revealed fundamental structural and mechanistic insight into the features that distinguish lipid scramblases from ion channels of the family. By combining data from X-ray crystallography and lipid transport experiments, our initial studies of the fungal lipid scramblases nhTMEM16 defined the architecture of a novel family of membrane proteins and the mechanism how the protein catalyzes lipid transport. In this structure, each subunit of the dimeric protein harbors a hydrophilic membrane-spanning furrow termed ‘subunit cavity’, which provides a pathway for the polar lipid headgroups to diffuse between the two leaflets of the bilayer whereas the hydrophobic lipid tail remains embedded in the membrane. In later studies by cryo-EM, we could show how the activity of nhTMEM16 is regulated by the binding of Ca2+ to a conserved site located within the membrane binding domain, which causes the opening of the ‘subunit cavity’ from its collapsed state in the Ca2+-free form. Combined with electrophysiology experiments, our cryo-EM studies of the ion channel TMEM16A revealed that the helices constituting the subunit cavity in a ‘lipid scramblase’ have rearranged in the channel TMEM16A to form an aqueous pathway for ions that is shielded from the membrane. TMEM16 channels are activated by an equivalent Ca2+-binding event as found for scramblases, which causes a rearrangement of a membrane-spanning α-helix contributing to the binding-site that couples to the narrow neck of the pore to open a gate. Due to its vicinity to the ion conduction path, the bound ligand contributes to permeation by altering the electrostatics of the pore. Finally, we were able to demonstrate that TMEM16F functions as both, a lipid scramblase and a non-selective ion channel and that both functions are activated via the same Ca2+-binding step. The structure of TMEM16F determined by cryo-EM closely resembles the TMEM16A structure thus suggesting that both functions are catalyzed by the same unit where the open cavity mediating lipid flow is in equilibrium with a collapsed ion conduction pore. Together, our experiments have revealed the common mechanistic basis for previously unknown transport processes and they have provided a foundation for the development of novel therapeutic strategies.