Bestrophins are a family of pentameric calcium-activated chloride channels that respond to intracellular calcium (Ca2+) by allowing the flow of monovalent anions across the membrane. Bestrophins are activated in the presence of nanomolar to micromolar concentrations of Ca2+ and exhibit a time- and concentration -dependent inactivation of current. The structural basis behind activation and subsequent inactivation in response to increasing Ca2+concentrations has not been fully elucidated at the molecular level. Bestrophins are expressed in a wide variety of tissues, and often within specialized cell types. Best1 is expressed in the retinal pigmented epithelial (RPE) cells of the eye and mutations in the BEST1 gene lead to a class of retinal degenerative diseases called bestrophinopathies. BEST2, on the other hand, is expressed in the basolateral membrane of the nonpigmented ciliary epithelial (NPE) cells of the eye, where it has been linked to generation of intraocular pressure, suggesting it may represent a pharmacological target to treat glaucoma. Sequence analysis of Best1-4 reveals Best2 has a difference in the cytosolic pore constriction, a region previously implicated in channel gating. The goal of this project is to 1) generate a structural model to explain Ca2-dependent activation and inactivation in a mammalian Best2 channel, including electrophysiological analysis and structural analysis of rationionally designed mutants to test the model, and 2) investigate the structural differences within the cytosolic constriction of Best2 that distinguish it from other bestrophins. Completion of these aims will inform on the function of bestrophins throughout the body, and will guide structure-based drug design to specifically target Best2 and not other bestrophins, which may have potential to treat glaucoma.
Bestrophins are a class of ion channels that are expressed in specific tissues, and Best2 is expressed in the eye, where it has been implicated as a therapeutic target to treat glaucoma. Bestrophins are activated by low levels of calcium and inactivated by high levels of calcium, and the proposed research will generate a structural model to explain how increasing calcium levels affect bestrophin conformation and will also identify structural differences between Best2 and Best1. The resulting structural data will be supported by electrophysiological experiments and structural analysis of mutants designed to test the proposed model, which will inform on structure-based drug design of therapeutics that can target Best2 and not other bestrophins, which may be important to reduce side effects.
