Although it was hypothesized some time ago that Best disease was caused by a defect in a chloride-selective ion channel, but only recently has this been established definitively. After human bestrophin-1 was cloned, Jeremy Nathan's lab expressed several bestrophin cDNAs in mammalian cells and found that they evoked novel chloride currents. Around the same time, we became interested in bestrophins while searching for proteins that we thought could be calcium-activated chloride channels. Since then, we have shown that bestrophins are indeed chloride channels that are activated by increases in intracellular free calcium. The most compelling evidence that bestrophins are chloride channels is that we can mutate different amino acids and alter the ability of the channel to distinguish between chloride and thiocyanate anions. This shows that bestrophin must be a component of the channel pore.

(1) Structure-function studies to determine how bestrophin channels work on the molecular level. We have made substantial progress in identifying the amino acid residues that play roles in forming the chloride-selective pore. In the model of mouse bestrophin-2 to the left, mutating red residues to cysteine kill channel function, whereas green residues behave as wild type. Residues of other colors have distinct phenotypes with regard to their ability to select among anions or to conduct anions in different directions across the membrane. We are also interested in how the channel is opened by calcium: Where is the Ca-binding site? Are other proteins involved? The cytoplasmic domains of the bestrophins are intriguing: although the first 300 amino acids are highly conserved from worms to human, the C-terminal third of the protein is very different among different bestrophins. The function of this region of the protein remains a mystery.

(2) Physiological function of bestrophin and other chloride channels in the eye. Human bestrophin-1 has been shown to be located in the basolateral membrane of the retinal pigment epithelium, a layer of pigmented cells at the back of the eye. These cells play an integral role in vision: they regenerate the visual pigment after it has been bleached by light, they phagocytose old photorecptor membranes, and they maintain the fluid and ionic balance of the extracellular fluid surrounding the photoreceptors. The mcirograph to the left shows an isolated retinal pigment epithelial cell. We are studying the ionic currents in these cells to understand the roles they play in retinal function.

(3) Function of other bestrophins. In mammals, there are 4 bestrophin genes. Although in humans, bestrophin-1 clearly has a role in the eye, the physiological function of the other 3 bestrophins is not known. In the nematode C. elegans, there are more than 25 different bestrophins. We wonder why. We are investigating the functions of bestrophins in knockout and transgenic mice.