Biological membranes define the boundaries of all cells and of their compartments. Ion channels, transporters and lipid scramblases are specialized membrane proteins that enable the movement of ions, nutrients and lipids in and out of cells. Our lab aims to elucidate the structural and mechanistic underpinnings of ion and lipid transport. To this end, we employ a wide range of approaches, from electrophysiological recordings, functional reconstitution of purified proteins, direct binding measurements and single particle cryo-electron microscopy. We focus on two families of membrane proteins, the CLC channels and transporters and the TMEM16 channels and scramblases. Members of these protein families regulate key processes in human physiology, such as muscle contraction, vesicular acidification, blood coagulation and membrane repair. Numerous inherited disorders are caused by mutations in genes encoding for CLC or TMEM16 proteins, further highlighting their importance in human health. Remarkably, these protein families display an unprecedented functional diversity: the CLCs are either passive chloride channels or proton-coupled secondary active transporters, while the TMEM16s function as Ca2+-gated ion channels, phospholipid scramblases or dual-function channel/scramblases. Thus, by studying these proteins we aim not only to understand fundamental aspects of human health, but also to exploit this unique diversity to elucidate how functional divergence can emerge from similar structural scaffolds.  

Structural bases of ion and lipid transport by the TMEM16s

The plasma membrane of all eukaryotic cells is asymmetric as active pumps externalize phosphatidylcholine and sequester phosphatidylserine to the inner leaflet. The rapid collapse of this asymmetry, externalizes phosphatidylserine to initiate signaling pathways involved in diverse physiological responses, leading to apoptosis, blood coagulation and membrane repair. The proteins mediating this collapse are called phospholipid scramblases. Remarkably, we found that the TMEM16s are a functionally divergent family of membrane proteins, where some are Cl-channels while the majority are dual function scramblases/non-selective ion channels. More recently, using a combination of cryo-electron microscopy, molecular dynamics simulations and functional assays we found how Ca2+binding induces opening of the lipid pathway to prepare them for function, and how their interaction with the surrounding membrane produces a distortion that enables the rapid movement of lipids between leaflets.

Recent publications: Malvezzi et al., PNAS, 2018 , Lee et al., Nat Comms, 2018 , Falzone et al., eLife, 2019 Collaborations: Weinstein lab , Carpenter lab , Di Lorenzo lab

Gating and selectivity in CLC channels and transporters 

The CLC channels and transporters form a widely distributed family of membrane proteins, which mediate anion transport. Mutations in six out of the nine human CLC genes cause genetic disorders of bone, kidney, brain and muscle. Although atomic resolution structures of both CLC functional subtypes are available, it is not clear what elements differentiate the channels from the exchangers or how these proteins rearrange in order to allow ion movement. We use a combination of atomic-scale mutagenesis, molecular dynamics simulations and functional recordings to elucidate the molecular bases of CLC gating and selectivity. To identify the molecular origin of the functional divergence of the CLC channels from the transporters we use statistical phylogenetics and evolutionary bionformatics combined with functional assays. These efforts will lead to a new molecular and conceptual framework to understand CLC function, and more broadly of the molecular determinants of active and passive transport. 

Recent publications: Vien et al., JGP, 2017 , Basilio et al., NSMB, 2014 Collaborations: Ahern lab , Tajkhorshid lab