Calcium activated Chloride Channels (CaCCs) and other TMEM16 family members form ion channels and/or lipid scramblases that help orchestrate a large number of cellular processes. Humans express 10 different paralogs labeled TMEM16A-K (skipping I) that are expressed throughout the body, and they aid in diverse phenomena including coagulation of the blood, suppression of inflammatory signals in the joints, control of pain through nociceptive neurons, and modulating neuronal excitability in multiple brain regions ? just to name a few. How this family can be involved in so many different physiological processes remains an intriguing open question. The founding member (TMEM16A) was cloned by 3 labs (including the Jan lab) in 2008 making it possible to elucidate the biological roles listed above, but also ushering in the ability to dissect the biophysical properties of these proteins. In the following years, the Jan lab employed mutagenesis screens, electrophysiology, and small molecule screening to uncover the ion conduction, lipid scrambling, and gating properties of TMEM16A and F in addition to solving high resolution cryo-EM structures (in collaboration with the Cheng lab) of TMEM16A (a Cl- channel) and structures of TMEM16F (a dual scramblase/ion channel). Meanwhile, the Grabe lab was the first to show in atomic detail how nhTMEM16 (a fungal scramblase) flips lipids by inducing large-scale deformations in the membrane that thin the bilayer near a hydrophilic grove that aids polar headgroups passing from one leaflet to the other. Despite these advances, fundamental questions about the function of these proteins remain that we intend to answer here. First, phosphatidylserine (PS) exposure to the outer leaflet of the plasma membrane via TMEM16F is the key signaling event that initiates platelet-dependent coagulation and microvesicle (MV) production; however, no one has demonstrated how a TMEM16 flips a negatively charged PS molecule at the atomic level under physiological conditions, the lipid specificity of TMEM16s is poorly understood, and it has been suggested that scramblases may also accomplish lipid flipping via an ?out of the groove? mode in addition to the one revealed by the Grabe lab. Second, we hypothesize that Cl- conduction occurs via a dedicated pore shielded from the membrane in Cl- selective CaCC, but despite the existence of many TMEM16A structures, this has not been shown. We also hypothesize that scramblases exhibit selectivity that is lipid-dependent because ions co-permeate with lipids at the protein-membrane interface. Together, our studies will reveal basic mechanisms related to how TMEM16 family members carry out a diverse set of biological phenomena.
Humans express 10 different TMEM16 family members that are expressed in a tissue specific manner, and through their action as ion channels and/or lipid scramblases, they have been implicated in normal physiology such as blood coagulation, nociception, and protection from inflammatory arthritis as well as diseases such as cancer. Unfortunately, it remains unclear how these proteins flip lipids, select one ion over another, and whether all members accomplish these tasks in the same manner. The mechanistic studies outlined here will attempt to answer these questions at the atomic level potentially revealing new therapeutic avenues to treat diseases ranging from stroke to cancer.