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

Abstract

Cytoplasmic calcium (Ca 2+ ) 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 Ca 2+ -independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca 2+ activation and ion selectivity. A "Ca 2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca 2+ . Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca 2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca 2+ -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 Ca 2+ 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.calcium-activated chloride channel | CaCC | ion selectivity | channel gating | ion channel biophysics E ukaryotic bestrophin proteins constitute a family of Ca 2+

Bestrophin 1 and retinal disease

“…The importance of these is exemplified by the fact that the I205T BEST1 mutation has been reported to cause retinitis pigmentosa (Davidson et al, 2009). Recent electrophysiological studies performed by Vaisey et al have revealed specific regions that control both ion selectivity and calcium activation (Vaisey et al, 2016). Their experiments strongly demonstrate that the neck does not significantly affect ion selectivity but instead primarily serves to act as a gate to calcium-dependently control chloride permeation.…”

“…Their experiments strongly demonstrate that the neck does not significantly affect ion selectivity but instead primarily serves to act as a gate to calcium-dependently control chloride permeation. Moreover, they find that the cytosolic aperture of the pore controls the relative permeability of the channel to different anions (Vaisey et al, 2016). …”

“…Although disease-causing BEST1 mutations exist throughout the entire Best1 protomer (http://www-huge.uni-regensburg.de/BEST1_database/home.php?select_db=BEST1), many mutations associated with the bestrophinopathies are prevalent in or around the first restriction site and the Ca 2+ clasp site. A fourth critical region may be the cytosolic aperture of the pore, which significantly affects relative permeabilities among anions (Vaisey et al, 2016). …”

The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

“…5) 22,23 . Intriguingly, the animal Bestrophin channel, whose narrow pathway is formed by three layers of Ile, Phe, and Phe, is an anion selective channel while the bacterial Bestrophin, which contains Ile, Ile, and Phe at the bottle neck, has been shown to be cation selective channel 22,24,25 .…”

Structural basis for ion selectivity in TMEM175 K+ channels

“…It is bound by eight water molecules in an anti-prismatic geometry ( fig. S8), which are in turn coordinated by backbone oxygens of Leu 42 , Ser 43 and Ser 44 (Fig. 2B).…”