Abstract
Human Bestrophin1 (hBest1) is a Ca2+-activated Cl− channel in retinal pigment epithelium (RPE) essential for retina physiology, and its mutation results in retinal degenerative diseases that have no available treatments. Here, we discover that hBest1’s channel activity in human RPE is significantly enhanced by adenosine triphosphate (ATP) in a dose-dependent manner. We further demonstrate a direct interaction between ATP and bestrophins, and map the ATP-binding motif on hBest1 to an intracellular loop adjacent to the channel activation gate. Importantly, a disease-causing mutation of hBest1 located within the ATP-binding motif, p.I201T, diminishes ATP-dependent activation of the channel in patient-derived RPE, while the corresponding mutants in bestrophin homologs display defective ATP binding and a conformational change in the ATP-binding motif. Taken together, our results identify ATP as a critical activator of bestrophins, and reveal the molecular mechanism of an hBest1 patient-specific mutation.
Highlights
Human Bestrophin[1] is a Ca2+-activated Cl− channel in retinal pigment epithelium (RPE) essential for retina physiology, and its mutation results in retinal degenerative diseases that have no available treatments
These results demonstrate a direct interaction between KpBest and adenosine triphosphate (ATP) without the requirement of ATP hydrolysis
To test whether ATP is an activator of hBest[1] under physiological conditions, we examined the influence of ATP on endogenous Ca2+-dependent Cl− current in human RPE cells
Summary
Human Bestrophin[1] (hBest1) is a Ca2+-activated Cl− channel in retinal pigment epithelium (RPE) essential for retina physiology, and its mutation results in retinal degenerative diseases that have no available treatments. Ca2+-independent activation has been reported for several bestrophin homologs, such as a purified bacterial bestrophin from Klebsiella pneumoniae (KpBest) measured in lipid bilayer[13], and human Bestrophin[2] (hBest2) and Drosophila Bestrophin[1] (dBest1) heterologously expressed in HEK293 cells[11]. These functional results suggest that bestrophins either remain constantly open or have additional activator(s) besides Ca2+ under physiological conditions. HBest1-mediated endogenous Ca2+-activated Cl− current in human induced pluripotent stem cell (iPSC) derived RPE (iPSC-RPE) displays ATP-dependent activation, while a disease-causing mutation I201T within the ATP-binding motif displays impaired ATP-dependent activation in patient-derived iPSC-RPE. Our results uncover ATP as an interacting activator of bestrophins and the molecular mechanism of a BEST1 patientspecific mutation
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