2022 Theses Doctoral

Structural studies on bestrophin anion channels by cryogenic electron microscopy

Owji, Aaron Paul

Bestrophins are a family of calcium (Ca²⁺) -activated chloride (Cl⁻) channels (CaCCs) with functional importance in eye physiology. Mutations to the VMD2 gene, which encodes the Best1 protein, cause an array of degenerative eye disorders called bestrophinopathies, which result from aberrant CaCC activity of the Best1 channel in the pigmented epithelium of the retina. While there are four bestrophin paralogs in mammals (Best1-4), the only current structures are of Best1 homologs. The structure of the prokaryotic homolog of Best1 from Klebsiella pneumonia (KpBest) was previously solved in this lab, representing the first structure of a Best1 homolog at the time.

This initial study laid the foundational groundwork in the field and contributed significant knowledge to understanding the bestrophin structure-function relationship. Nevertheless, significant questions remain regarding bestrophin function, such as the molecular determinants underlying its Ca²⁺-dependent gating and anion selectivity. This dissertation uses single-particle cryogenic electron microscopy paired with electrophysiology to probe the structure-function relationship of mammalian bestrophins under different buffer conditions and reveals conformational dynamics involved in gating of wild-type channels. Key regions of the channel contributing to its function are described at the atomic level leading to development of a gating model to explain Ca²⁺-dependent activation and inactivation in mammalian bestrophins.