Many physiological events, such as heart beat and muscle contraction, are orchestrated by the cellular concentrations of certain ions, which flow in and out of the cell through designated structures on the cell membrane called ion channels. Dysregulation of ion channel/flux is clinically linked to many diseases. The human bestrophin-1 (hBest1) is a chloride (Cl-) conducting channel highly expressed in eyes. So far, over 120 distinct mutations of hBest1 have been identified to associate with multiple kinds of eye diseases that cause vision loss. Although many hBest1 disease-causing mutants have been documented as defective Cl- channels, how this defect pathologically results in eye disorders remains elusive. Moreover, it is largely unclear how the Cl- channel function of hBest1 is regulated/activated. Therefore, it is of both biological and biomedical significance to thoroughly understand the structure and function of hBest1. The goal of this proposal is to elucidate the working mechanisms of hBest1 by crystallography and electrophysiology from the structural and functional aspects, respectively. Importantly, a recently obtained structure model of a bacterial bestrophin homolog (KpBest, from Klebsiella pneumoniae) provides novel clues and a structural basis for the research aims in this proposal. To be specific, in Aim 1, the two putative ion permeation gates on bestrophins, as suggested by the KpBest structure model, will be examined in both KpBest and hBest1;
in Aim 2, the hypothesis that ATP directly interacts and activates bestrophins will be tested by co-crystallizing ATP and KpBest, and further functionally examined with both KpBest and hBest1;
in Aim 3, the crystal structure of eukaryotic bestrophin(s) and/or hBest1 will be obtained using the KpBest structure as a search model. During the K99 phase (Aim 1, and parts of Aims 2 and 3), I will be mentored by Dr. Wayne Hendrickson, a leader in the field of protein crystallography. This work will shed new light on how disease-causing hBest1 mutations influence the structure and function of the channel to contribute to eye diseases, and provide valuable insight for structure-based specific channel activator/inhibitor design in the future.
Ion channels control the ion flux in and out of the cells, and thus play an essential role in physiology and pathology. Human bestrophin-1 (hBest1) resides on the retina to specifically traffic chloride, and its dysfunction by mutations has been linked to eye diseases featured by vision loss. This proposal is aimed to understand the structure and working mechanisms of hBest1 chloride channel, and the results will provide novel knowledge and principles for both basic research and biomedical applications.
|Kittredge, Alec; Ward, Nancy; Hopiavuori, Austin et al. (2018) Expression and Purification of Mammalian Bestrophin Ion Channels. J Vis Exp :|
|Kittredge, Alec; Ji, Changyi; Zhang, Yu et al. (2018) Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations. J Vis Exp :|
|Zhang, Yu; Kittredge, Alec; Ward, Nancy et al. (2018) ATP activates bestrophin ion channels through direct interaction. Nat Commun 9:3126|
|Yang, Tingting; Justus, Sally; Li, Yao et al. (2015) BEST1: the Best Target for Gene and Cell Therapies. Mol Ther 23:1805-9|