2026 Theses Doctoral

Structure, Function and Regulation of the Bestrophin Family of Calcium activated Anion Channels

Kittredge, Alec

Bestrophins are a family of calcium-activated chloride channels (CaCCs) with relevance to human physiology and a myriad of eye diseases termed “bestrophinopathies”. In humans, Bestrophin1 (Best1) is expressed on the basolateral side of the retinal pigment epithelium (RPE), and its dysfunction due to Best1 mutations leads to retinal degeneration, causing loss of sight and potentially blindness in patients. Best2 is expressed in the ciliary body of the eyes, Best3 is expressed at low levels throughout the body, and Best4 is primarily expressed in a subset of colonic cells. Since the identification of bestrophins as CaCCs nearly two decades ago and the subsequent identification of Best1 as the CaCC responsible for chloride permeation in the RPE, extensive studies from electrophysiological and structural biology perspectives have sought to define bestrophin proteins’ key features including calcium sensing, gating, inactivation, anion selectivity.

Previous X-ray crystallography structures from the prokaryotic homolog of Best1, Klebsiella pneumoniae (KpBest), the Best1 homolog from Gallus gallus (chicken Best1, cBest1), and cryogenic electron microscopy (cryo-EM) structure of Best2 from Bos taurus (bovine Best2, bBest2) identified two landmark channel constrictions, namely the highly conserved “neck” restriction of the ion conduction pathway at the level of the inner leaflet of the plasma membrane, and the divergent “aperture” at the cytosolic exit. Meanwhile, whole-cell patch clamp, planar lipid bilayer, and other electrophysiologic analyses using these models as well as the human Best1 have provided ample evidence describing the functional properties of the bestrophin channels.

Yet, at the start of this work, no human bestrophin structures had yet to be solved. Thus, we solved the structures of each of the human bestrophins and elucidated aspects of the channels such as their open/closed probabilities, ion conduction pathway diameters, autoregulatory mechanisms, and determined their intermediate or partial states, which explain their distinct electrophysiologic profiles. Furthermore, while elucidating the structures of bestrophins is critical in uncovering how they function, proteins do not exist in a vacuum – there are other proteins, small molecules, lipids, etc. with which they interact.

For instance, the identification of Best1 in the brain and its role in glutamate and γ-aminobutyric acid (GABA) transport in astrocytes is a newer topic of research, only probed within the past decade. How bestrophin channels interact with their environment – to both be influenced and to influence the function of other proteins and small molecules in their physiologic environment – is a largely unexplored area of research.

As is explained here, we identified two protein binding partners of bestrophins – glutamine synthetase (GS) interacts with Best2 with direct physiologic impact in the nonpigmented epithelium of the ciliary body, and glutamate decarboxylase (GAD) interacts with Best1 with relevance in the RPE. Both of these enzymes are involved in the metabolism of critical neurotransmitters glutamate and GABA, and functional studies suggested that both glutamate and GABA can not only pass through but also potentially interact with the bestrophin channels.

We then tested and confirmed direct interactions between these compounds with bestrophins proteins, obtaining multiple small molecule-bound bestrophins structures with altered open probabilities compared to the apo structures. We identified chemical analogs with functional and structural influences on the channels, and confirmed their effect for restoring Best1 function in cells bearing autosomal dominant, loss-of-function disease-causing Best1 mutations back to wild type-like. As there are currently no specific Best1-targeting drugs nor treatment options for bestrophinopathies, this has great biopharmaceutical potential.