Featured Article - April 2015
Short description: Crystal structures of bestrophin ion channels unravel the molecular basis of retinopathies.
Bestrophins are calcium-activated chloride channels that are broadly distributed in metazoan tissues and especially abundant in the retinal pigment epithelium. Four human bestrophins (BEST1–4) are known and sequence-related prokaryotic proteins have been identified. While the physiological roles of bestrophins have yet to be fully determined, mutations of human BEST1 are linked to retinopathies such as vitelliform macular dystrophy, or Best disease. To shed light on bestrophin structure and function, Hendrickson and colleagues (PSI NYCOMPS) have solved the crystal structure of a bacterial ortholog of bestrophin from Klebsiella pneumoniae (KpBest, PDB 4WD8) at 2.3-Å resolution, and Long and colleagues have determined the structure of chicken BEST1 (PDB 4RDQ) at 2.85-Å resolution.
Both the eukaryotic and prokaryotic channels are homopentameric (thus clarifying bestrophin stoichiometry) and share a unique architecture consisting of a transmembrane region to which each protomer contributes four predominantly alpha-helical segments, and a large cytoplasmic region, also mostly alpha helical. The ion-conducting pore spans the entire length of BEST1: it begins at the extracellular side with a funnel-shaped vestibule that narrows midway through the transmembrane region to form a ∼6 Å-wide neck spanning three turns of the α2 helix, lined by hydrophobic residues (isoleucines and phenylalanines). Following the neck, the pore opens to form a wide inner cavity that spans the cytoplasmic region.
Long and colleagues reconstituted calcium-activated chloride channel function using purified BEST1 protein. From their structural work, they identified five symmetrical Ca2+-binding sites, each formed by acidic residues clustered at the interface between the transmembrane and cytoplasmic regions. The proximity of the Ca2+-binding sites to the neck, which likely constitutes the channel gate, suggests a possible mechanism whereby Ca2+ binding induces a structural rearrangement of the neck aperture, resulting in channel opening.
Hendrickson and colleagues found that, in contrast to eukaryotic bestrophins, KpBest conducts monovalent cations and does not require Ca2+ for activation. Using mutagenesis and electrophysiology analyses, they showed that the pore-lining hydrophobic residues in the neck affect ion selectivity. In particular, mutating Ile66 to the corresponding phenylalanine conserved in eukaryotic BEST1 channels makes KpBest permeable to anions. Long and colleagues identified anion-binding sites, two in the extracellular vestibule and one in the cytoplasmic cavity of each protomer, which may contribute to anion selectivity in eukaryotic bestrophins.
Finally, the mutations in human BEST1 associated with retinopathies are found to occur especially in the Ca2+-binding sites and the pore neck. Hence, these studies suggest that the mutations cause eye disease by altering channel gating and anion permeation.