Interacting Partners of Bestrophin Channels

Project Summary/Abstract The bestrophin proteins are a family of Ca2+-activated anion channels widely distributed from bacteria to mammals, with representatives in most metazoans including four in humans (Best1-4). They open in response to increases of intracellular Ca2+ to mediate the passive flow of Cl− and other anions. Best1 and Best2 both have critical roles in the eye. Best1 is predominantly expressed in retinal pigment epithelium (RPE) of the retina playing an essential role in generating a vision-related electrical signal named “light peak”, while mutations in the human BEST1 gene have been genetically linked to at least five retinal degenerative disorders collectively known as bestrophinopathies. Best2 is highly expressed in non-pigmented epithelium (NPE) of the ciliary body, and is involved in aqueous humor formation and drainage, which ultimately determine intra-ocular pressure (IOP). Knockout of Best2 in mice leads to a reduction of IOP, suggesting the pharmaceutical potential of targeting Best2 and its regulators for relieving ocular hypertension, which is a common risk factor for numerous eye diseases including open-angle glaucoma. However, despite bestrophins’ biological and pharmaceutical significance, little is known about their cellular regulation in a physiological context. More specifically, in a given cell/tissue, how is the bestrophin channel modulated to conduct which anion(s) for what downstream purpose(s)? The answer to this question lies within the protein network that interacts with bestrophins in different tissues/cells. Although ample evidence suggest the existence of tissue-specific interacting regulators of bestrophins, no protein is known to interact with Best2, while only a few have been reported to interact with Best1. Our lab studies the biophysics and regulation of bestrophin channels using a multidisciplinary platform consisting of cryogenic electron microscopy (cryo-EM), mass spectrometry, electrophysiological recording, CRISPR/Cas9-mediated genome editing, and stem cell reprogramming/differentiation. The goal of the next five years is to identify Best1 and Best2 interacting protein complexes in RPE and NPE, respectively, and to understand the functional consequences of these interactions. Overall, the proposed work will make contributions to multiple fields of research including calcium signaling, ion transport, membrane protein structure and ocular physiology, and the pipelines established in this work can be generally applied to study different ion channels and other membrane proteins of interest.