Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Abstract

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl− ion channels that are activated by intracellular Ca2+. At present, the structures and mechanisms responsible for Ca2+ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca2+ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca2+ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca2+ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca2+. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca2+ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca2+-binding loop (312–323) reduced the apparent Ca2+ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca2+-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca2+ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca2+ and suggest a model in which Ca2+ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ∼100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca2+ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca2+.

Stoichiometry and specific assembly of Best ion channels

“…6A) (18,26), because of the much smaller currents of hBest3 (1,15). To confirm that the quenching was due to iodide conduction through hBest, we either introduced BMD-causing mutations (W93C or R218C) that are known to block ion conduction through hBest1 (7,11) or deleted the Ca 2+ -binding domain at the C terminus (Δ299-304) that is needed for the activation of hBest1 (24). As expected, we observed no ATP-triggered quenching in either of the mutants (Fig.…”

“…Best1 is activated by Ca 2+ with a K d of ∼150 nM (24). Several pieces of evidence suggest that this activation is due to direct binding of Ca 2+ (25,26) to an EF hand located immediately after S6 (24).…”

“…Several pieces of evidence suggest that this activation is due to direct binding of Ca 2+ (25,26) to an EF hand located immediately after S6 (24). It is unclear how Ca 2+ -binding gates the channel and whether the EF hand is part of the gate or communicates with it.…”

Phasing and structure of bestrophin-1: a case study in the use of heavy-atom cluster compounds with multi-subunit transmembrane proteins

“…Early screening involved cloning of both fulllength and truncated forms of each isoform of the protein, where the truncated candidates were designed to end at the equivalent of human Best1 residue 398. This residue was selected in part owing to a report that calcium binding suffered if the protein was shorter than residues 1-380 (human Best1 numbering; Xiao et al, 2008), and the sequence conservation was increasingly weak beyond residue 398. Protein that was truncated at this point was shown to be active using an assay of purified, reconstituted protein (Kane Dickson et al, 2014).…”

“…Mutations in bestrophins lead to retinopathies owing to a dysregulation of chloride in the retinal pigment epithelium (Marquardt et al, 1998;Petrukhin et al, 1998;Davidson et al, 2009). The channel has a high affinity for calcium (K d of $150 nM) and has been proposed to have at least two domains of its primary sequence devoted to calcium binding, including a conserved stretch of ISSN 2059-7983 highly acidic amino acids (Qu et al, 2007;Xiao et al, 2008;Kranjc et al, 2009).…”