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ANOBEST Report Summary

Project ID: 339116
Funded under: FP7-IDEAS-ERC
Country: Switzerland

Mid-Term Report Summary - ANOBEST (Structure function and pharmacology of calcium-activated chloride channels: Anoctamins and Bestrophins)

Calcium activated chloride channels (CaCCs) constitute a diverse class of anion channels that are activated by an increase of the intracellular calcium concentration. These proteins play an important role in different physiological processes ranging from epithelial transport to the control of electrical excitability. Although they have been studied for decades, their molecular identity was revealed only few years ago, when members of the unrelated TMEM16/Anoctamin and Bestophin families were identified to function as CaCCs. The structural and functional characterization of both families is subject of the ERC funded project AnoBest. By now, our investigations have already resulted in a breakthrough in the understanding of the TMEM16 family. After the identification of TMEM16A as CaCC, it was initially anticipated that all members of the conserved TMEM16/Anoctamin family would function as ion channels. This initial assumption was proven wrong, when the related family member TMEM16F was found to be the long sought-after lipid scramblase in blood platelets, which plays an important role in the initiation of blood coagulation by catalyzing the calcium-triggered disruption of the membrane asymmetry. It is now believed that the majority of TMEM16 proteins function as lipid scramblases. In the course of the AnoBest project, we have successfully overexpressed and purified different members of this eukaryotic membrane transport family. In one case, for the fungal homolog nhTMEM16A, we have determined a crystal structure at high resolution, which has revealed a novel protein architecture that is representative for both branches of the family. In complementary functional assays, we have shown that nhTMEM16 functions as lipid scramblase and not as ion channel. nhTMEM16 has provided insight into potential mechanisms of lipid scrambling. During this process, the lipid head groups would diffuse along an exposed polar cavity of the protein on their way across the lipid bilayer while their hydrophobic tails remain in their favorable membrane environment. The nhTMEM16 structure has also allowed for the identification of a conserved calcium-binding site that underlies activation in channels and scramblases. We have subsequently used patch-clamp electrophysiology to show that, similar to scramblases, the chloride channel TMEM16A contains two ion conduction pores that are each contained within a single subunit of the dimeric protein and that are independently activated by Ca2+. Our work has thus provided important insight into a protein family with an unusual functional breadth. Ongoing studies employ different methods to elucidate the basis for the functional divide between channels and scramblases and the common mechanisms of Ca2+ activation.

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