The long-term objective of this proposal is to understand the mechanisms of ion permeation, ion selectivity, and gating in the bestrophin family of calcium-activated chloride channels. Mutations in bestrophin channels are correlated with an increasing number of inherited retinal degenerative diseases (bestrophinopathies) including a juvenile-onset form of retinal degeneration (Best vitelliform macular dystrophy). Humans have four bestrophin proteins (Best1-4). Best1, which is the primary focus of this research, is highly expressed in the retinal pigment epithelium (RPE) and associated with the majority of bestrophinopathies. Best1 channels form anion selective pores that are regulated by changes in the intracellular calcium concentration, by phosphorylation, and by changes in cell volume. Currently, three-dimensional structural information is not available for bestrophin or for any other calcium-activated chloride channel. Aside from being integral membrane proteins, bestrophin channels have no significant amino acid sequence homology to other known ions channels, suggesting that bestrophin channels have marked differences in mechanisms of ion selectivity, permeation and gating in comparison with other channel families. This study proposes to use X-ray crystallography to determine atomic structures of bestrophin channels. Biochemical and biophysical techniques, including an assay to measure ion channel activity in vitro, will be used to correlate channel function with structural analysis. With these approaches we aim to: 1) determine the 3-dimensional structures of bestrophin, 2) investigate the mechanism of anion selectivity, 3) study how the channel is gated by changes in intracellular calcium levels and modulated by phosphorylation, and 4) discern the molecular basis for bestrophinopathies. The research will reveal basic principles of bestrophin channel function, thereby making significant contributions to multiple fields of research including calcium signaling, ion channels, and the structural biology of eukaryotic membrane proteins. It will bring to light the molecular underpinnings of certain retinal diseases and will serve as a foundation to develop tools to further understand the physiology and dysfunction of bestrophin channels.
This proposal seeks to understand the architecture and molecular mechanisms of bestrophin, an ion channel, at an atomic level of detail. Mutations in bestrophin are associated with eye diseases including a juvenile-onset form of macular degeneration (Best vitelliform macular dystrophy) and other disorders of the retina. With the proposed studies we will discover the inner workings of the bestrophin channel and more fully understand the causes of these eye diseases.
|Vaisey, George; Miller, Alexandria N; Long, Stephen B (2016) Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel. Proc Natl Acad Sci U S A 113:E7399-E7408|
|Kane Dickson, Veronica; Pedi, Leanne; Long, Stephen B (2014) Structure and insights into the function of a Ca(2+)-activated Cl(-) channel. Nature 516:213-8|