Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel.
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.
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.|Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
George
G
Vaisey
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065.
Alexandria N
AN
Miller
Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; longs@mskcc.org.
Stephen B
SB
Long
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D000075002:Bestrophins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation
D000075002:Bestrophins; D018118:Chloride Channels; D000409:Alanine; D002118:Calcium
CaCC; calcium-activated chloride channel; channel gating; ion channel biophysics; ion selectivity