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Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.

ABSTRACT: Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based 'disease-in-a-dish' approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

SUBMITTER: Li Y

PROVIDER: S-EPMC5655127 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.

Li Yao Y Zhang Yu Y Xu Yu Y Kittredge Alec A Ward Nancy N Chen Shoudeng S Tsang Stephen H SH Yang Tingting T

eLife 20171024

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlat ...[more]

PMID: 29063836

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Project description:Elevation of extracellular Ca(2+) concentration induces intracellular Ca(2+) signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca(2+) pathways, but the direct mechanism responsible for the rise of intracellular Ca(2+) concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca(2+) signaling in frog parathyroid cells and show that Ca(2+)-activated Cl(-) channels are activated by intracellular Ca(2+) increase through an inositol 1,4,5-trisphophate (IP(3))-independent pathway. High extracellular Ca(2+) induced an outwardly-rectifying conductance in a dose-dependent manner (EC(50) ?6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca(2+)-induced and Ca(2+) dialysis-induced currents reversed at the equilibrium potential of Cl(-) and were inhibited by niflumic acid (a specific blocker of Ca(2+)-activated Cl(-) channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca(2+)-induced current, suggesting the change of intracellular Cl(-) concentration in a few minutes. Extracellular Ca(2+)-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca(2+)-induced current. IP(3) dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca(2+)-induced conductance. These results indicate that high extracellular Ca(2+) raises intracellular Ca(2+) concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl(-) conductance.

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Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.

Publications

Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca<sup>2+</sup>-dependent Cl<sup>-</sup> currents in human RPE.

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