The Road not Taken - Lipid/Ion Conduction Pathways in TMEM16 Protein Family

Abstract

Despite high sequence and structural similarity, members in transmembrane protein 16 family (TMEM16) can transport distinct cargoes cross cell membranes. TMEM16A is a calcium-activated chloride channel (CaCCs), which is critical in regulating neural excitability, nociception, smooth muscle contraction, secretion and gut motility, whereas TMEM16F is an enigmatic calcium-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. However, recent structures TMEM16A and TMEM16F fail to reveal conductive ion or lipid permeation pathways even in the presence of saturating calcium. Combining experiment and simulation, we have previously identified a conserved inner activation gate consisting of three hydrophobic residues, where a single Lys mutation can convert both TMEM16A L543K and TMEM16F F518K into constitutively active scramblase. We have also identified a specific PIP2 binding site that regulates TMEM16A desensitization. Here, we report direct observations of the close to open transitions of the permeation pathway in the calcium bound TMEM16F F518K mutant as well wild-type TMEM16A in the presence of PIP2. Direct observations of the conductive states of TMEM16A and TMEM16F in our structural models thus give us a unique opportunity to dissect TMEM16 ion and lipid permeation pathways and understand their molecular gating mechanisms. Our findings will shine lights on understanding the physiological functions of TMEM16-mediated ion and lipid transport.