2016 - Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel
Port-a-Patch publication in Proceedings of the National Academy of Sciences of the United States of America (2016)
Vaisey G., Miller A.N., Long S.B.
PNAS (2016) 103(47):e7399-e7408
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.
BEST1 is a Ca2+-activated chloride channel found in a variety of cell types that allows chloride to traverse the plasma membrane. Mutations in BEST1 can cause macular degeneration. The mechanisms for anion selectivity and Ca2+-dependent activation of BEST1 are unknown. Here, we show that a hydrophobic “neck” region of the channel’s pore does not play a major role in ion selectivity but acts as an effective gate, responding to Ca2+ binding at a cytosolic sensor. Mutation of a cytosolic “aperture” dramatically affects relative permeabilities among anions. These insights help rationalize how disease-causing mutations in BEST1 affect channel behavior and contribute to a broader understanding of ion channel gating and selectivity mechanisms.