Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 113; no. 47; pp. E7399 - E7408
• Main Authors: Vaisey, George, Miller, Alexandria N., Long, Stephen B.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22-11-2016
• Series: PNAS Plus
• Subjects: Amino acids; Biological Sciences; CaCC; Calcium; calcium-activated chloride channel; channel gating; Cytoplasm; INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; ion channel biophysics; ion selectivity; Ions; Mutation; PNAS Plus; ion channel biophysics; CaCC; ion selectivity; channel gating; calcium-activated chloride channel
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1614688113

Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A “Ca2+ clasp” within the channel’s intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic “neck” of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a “gate” that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel’s ion selectivity. We find that mutation of a cytosolic “aperture” of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single “selectivity filter,” in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.