Structures and gating mechanisms of human bestrophin anion channels

Abstract

Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of calcium (Ca2+)-activated chloride (Cl−) channels with critical involvement in ocular physiology and direct pathological relevance. Here, we report cryo-EM structures of wild-type human Best1 and Best2 in various states at up to 1.8 Å resolution. Ca2+-bound Best1 structures illustrate partially open conformations at the two Ca2+-dependent gates of the channels, in contrast to the fully open conformations observed in Ca2+-bound Best2, which is in accord with the significantly smaller currents conducted by Best1 in electrophysiological recordings. Comparison of the closed and open states reveals a C-terminal auto-inhibitory segment (AS), which constricts the channel concentrically by wrapping around the channel periphery in an inter-protomer manner and must be released to allow channel opening. Our results demonstrate that removing the AS from Best1 and Best2 results in truncation mutants with similar activities, while swapping the AS between Best1 and Best2 results in chimeric mutants with swapped activities, underlying a key role of the AS in determining paralog specificity among bestrophins.

Cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels in the HEK293 cells

“…3A ). First, the functional consequences of hBEST1 345 (hBEST1 1-345 ) mutant, which encodes only transmembrane core domain ( 14 ), were accessed by the electrophysiological recordings. The hBEST1 345 showed robust [Ca 2+ ] i -dependent currents with the EC 50 of ∼470 nM (3-8 observations), which differs less than an order of magnitude (∼2.5-fold decrease) to wildtype hBEST1.…”

“…Structural studies have revealed the three-dimensional architectures of several BEST channel homologs including chicken BEST1 (cBEST1) ( 11 , 12 ), bovine BEST2 (bBEST2) ( 13 ), hBEST1, and hBEST2 ( 14 ). The structures showed that the functional BESTs are formed by the homo-pentameric assembly.…”

“…The neck is formed by the pore-lining hydrophobic residues ( 76 Ile, 80 Phe, 84 Phe) and the aperture is formed by the nonpolar residue ( 205 Ile in hBEST1). Third, the auto-inhibitory segment (AS, residue 346-378 in hBEST1) wraps the periphery of the BEST channel cytosolic domain in the closed state, but the detachment of AS may facilitate channel opening ( 14 ). However, none of the BEST channel structures visualized the C-terminal domain beyond amino acid residue 379.…”

“…The structures of cBEST1 and bBEST2 were determined by using the C-terminal truncated proteins (cBEST 1-405 , cBEST1 1-345 , or bBEST2 1-406 ; the numbers indicate the amino acid residues used in the structural studies) for the ease of structural determination ( 11 - 13 ). However, the full-length hBEST1 and hBEST2 structures also failed to resolve the C-terminal structure ( 14 ), which might be due to the disordered C-terminal domain in the cryo-EM conditions or intrinsically disordered nature. Thus, the structural role of the cytosolic C-terminal domain remains elusive.…”

Influence of Electronic Polarization on the Binding of Anions to a Chloride-Pumping Rhodopsin

“…Structures of future interest might include the Na + /I − symporter (NIS) (PDB: 7UV0) that mediates active I − transport and contains a highly conserved I − binding site consisting of predominantly hydrophobic and aromatic residues (15). Similarly, the Cl − -selective human bestrophin channels (hBest1& hBest2) contain a set of highly conserved hydrophobic residues within the neck of the permeation pathway (42). These structures suggest not only an important role for hydrophobic contacts in the ion permeation pathway but potentially influential roles of anion-π interactions.…”