Ion and Lipid Transport in the TMEM16 and OSCA/TMEM63 Families

Abstract

Ions and lipids are asymmetrically distributed across nearly all biological membranes, with asymmetry being destroyed through passive transport by the respective actions of membrane-resident ion channels and lipid scramblases. Uniquely, the TMEM16 family contains both calcium-activated ion channels and channel/scramblases, blending seemingly disparate functions within a conserved ten-transmembrane helical architecture. The TMEM16 family is also evolutionarily related to the OSCA/TMEM63 and TMC families of mechanically activated ion channels, forming the Transmembrane Channel/Scramblase (TCS) superfamily. Mammalian members of the TCS superfamily play critical roles in wide-ranging processes such as auditory, olfactory, visual, thirst, and pain sensation, muscle contraction, hemostasis, reproduction, and viral infection, whereas pathogenic variants are associated with bleeding disorders, movement disorders, epilepsies, developmental deficits, sensory defects, and cancers. Yet, given their shared ten-transmembrane helical architectural, it is unclear what molecular mechanisms exist among members of this family to discriminate between ion and ion/lipid substrates. Moreover, the extent of lipid permeability across the TCS is unknown but supremely important to understand their physiological and pathophysiological roles.This dissertation addresses these knowledge gaps using a combination of patch clamp electrophysiology and lipid scramblase assays in heterologous overexpression systems, with supporting evidence provided collaboratively through structural and computational approaches. Chapter 2 provides broad evidence that lipid permeability is an innate feature across the superfamily, endowed by the shared ten-transmembrane helical architecture. This chapter demonstrates that single mutations in TMEM16F, TMEM16A, OSCA1.2, TMEM63A, and TMEM63B confer constitutive lipid permeability by disrupting a key interface along the substrate permeation pathway. This effect also transcends the differential mechanisms of activation (i.e., calcium versus mechanical activation) across the TMEM16 and OSCA/TMEM63 families and implies that lipid permeability may also be a shared feature among members of the TMC family. Chapter 3 extends these findings by uncovering ion permeation mechanisms for a single member of the OSCA/TMEM63 family, OSCA1.2. This focused effort reveals mutations in strongly conserved residues within the OSCA/TMEM63 family that promote or prevent mechanically activated ion conduction. Together with Chapter 2, these efforts establish a framework to evaluate clinical variants in TMEM63B in Chapter 4. These variants are associated with neurological and hematological abnormalities, including developmental and epileptic encephalopathies and severe transfusion-dependent anemias. Remarkably, like the mutations identified in Chapter 2, TMEM63B clinical variants near the substrate permeation pathway exhibit constitutive lipid permeability, which may contribute to their underlying pathophysiology. Mutations identified in Chapter 3 that prevent ion permeation in OSCA1.2 also prevent TMEM63B ion and variant-induced lipid permeability, underscoring the translatable mechanisms identified across these works. In summary, this dissertation represents a contribution that critically alters how ion and lipid transport are viewed among the TMEM16 and OSCA/TMEM63 families, and across the TCS superfamily more broadly, connecting molecular mechanisms to health and disease.