1.A.46 The Anion Channel-forming Bestrophin (Bestrophin) Family

The bestrophins comprise a large family of Ca²⁺-regulated homo-tetrameric anion (chloride and bicarbonate; Qu et al., 2006; Bharill et al., 2014) channels found in animals, plants, fungi and bacteria (Sun et al., 2002; Hagen et al., 2005). Four bestrophins (VMD1-4) are encoded in the human genome; 4 in Drosophila melanogaster and 24 in Caenorhabditis elegans. Bestrophin-1 (VMD2 gene product) is the autosomal dominant vitelliform macular dystrophy protein or the Best disease protein (Burgess et al., 2008; Yu et al., 2008). The genetic defect causes loss of central vision and defects in the retinal pigment epithelium. The 585 aa VMD2 exhibits two strongly hydrophobic peaks in the N-terminal 100 residues, two moderately hydrophobic peaks centered at residues 150 and 200, respectively, and two more strongly hydrophobic peaks between residues 220 and 300. The remainder of the protein is strongly hydrophilic. Analyses suggest that the N- and C-termini are in the cytoplasm with three extracytoplasmic loops, suggesting a 4 TMS topological model with the moderately hydrophobic regions possibly dipping into the membrane (Tsunenari et al., 2003). hBest1 and sphingomyelin are miscible in surface films, and this property is a prerequisite for interaction with membrane domains (Mladenov et al. 2020). The human BEST paralogs (BEST1 - BEST4) are homologous in the N-terminal region, which forms the transmembrane helicases and contains the direct calcium-binding site, Ca²⁺-clasp. The cytosolic domain regulates the calcium sensitivity and surface expression of BEST1 channels (Kim et al. 2023).

Using single-molecule subunit analysis, Bharill et al. 2014 found that human Best1, 2, 3 and 4 each preferentially self-assembles into a homotetrameric channel. Thus, despite considerable conservation among human Bests, hBest1 has little or no interaction with other hBests or mTMEM16A. The domain responsible for assembly specificity was identified, and it also plays a role in channel function. Several human diseases are due to mutations in the members of this family (Stöhr et al. 2005).

Bestrophins can function both as Cl^- channels and as regulators of voltage-gated Ca²⁺ channels. The founding member, human bestrophin-1 (hBest1), is responsible for a dominantly inherited, juvenile-onset form of macular degeneration called Best vitelliform macular dystrophy. Mutations in hBest1 have also been associated with a small fraction of adult-onset macular dystrophies. It is proposed that dysfunction of bestrophin results in abnormal fluid and ion transport by the retinal pigment epithelium, resulting in a weakened interface between the retinal pigment epithelium and photoreceptors (Hartzell et al., 2008). There is compelling evidence that bestrophins are Cl^- channels, but bestrophins remain enigmatic because it is not clear that the Cl^- channel function can explain Best disease. In addition to functioning as a Cl^- channel, hBest1 also regulates voltage-gated Ca²⁺ channels. Some bestrophins are activated by increases in intracellular Ca²⁺ concentration, but whether bestrophins are the molecular counterpart of Ca²⁺-activated Cl^- channels remains in doubt. Bestrophins are also regulated by cell volume and may be members of the volume-regulated anion channel family (Hartzell et al., 2008).

The best disease-linked Cl^- channel hBest1 regulates Ca V 1 (L-type) Ca²⁺ channels via src-homology-binding domains (Yu et al., 2008). It probably interacts with the Ca(V)beta subunit, altering channel availability. This novel function provides a possible mechanism for the role of hBest1 in macular degeneration (Yu et al., 2008). Bestrophin is activated by Ca²⁺ which interacts with a C-terminal domain (EF1) to gate the channel. 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 approximately 100 disease-causing mutations in hBest1 are located in this region, the Ca²⁺ sensing domain (Xiao et al., 2008). Human disease-causing mutations in the N- and C-termini of hBest1 disrupt an N-C-terminal interaction and channel function of bestrophin 1 (Qu et al., 2009).

The Best1 channel activity is regulated by ceramide and phosphorylation. Protein kinase C (PKC) phosphorylates (serine 358) in hBest1 for sustained channel function. Channel activity is maintained by PKC activators, protein phosphatase inhibitors, or pseudo-phosphorylation by substitution of glutamic acid for serine 358 ()Xiao et al., 2009. Ceramide accumulation during early stages of retinopathy inhibits hBest1 function, leading to abnormal fluid transport across the retina, and enhanced inflammation.

Multiple members of the Bestrophin family are found in mammals, insects and C. elegans. At least twenty paralogues are encoded in the C. elegans genome. Different functionally characterized bestrophins (2 from humans, one from Drosophila and one from C. elegans) produce chloride conductances with distinctive I-V relationships and ion selectivities (Hagen et al., 2005). The ion selectivity of VMD1 is; SCN^-≥I^-≥NO₃^->Br^->Cl^->HCO₃^- (Yu et al., 2006).

Some bestrophins are much larger (~900 residues) than VMD2, while some are much smaller (~280 residues). For example, a C. elegans homologue (Q17851) is 884 residues long and exhibits repeated elements as well as at least 8 strongly hydrophobic putative TMSs. Each of the 4 pairs of TMSs are centered at positions 60, 150, 400 and 700 in the protein. Distant homologues in E. coli (YneE; P76146) and Salmonella typhi (Q8Z706) are 315-321 aas long and exhibit a hydropathy plot similar to the first 300 residues of VMD2. Thus, the proteins in the Bestrophin family are not uniform in size or topology (Hagen et al., 2005). 

Bestrophin calcium-activated chloride channels (CaCCs) regulate the flow of chloride and other monovalent anions across cellular membranes in response to intracellular calcium (Ca²⁺) levels. Mutations in bestrophin 1 (BEST1) cause certain eye diseases. Kane Dickson et al. 2014 presented X-ray structures of chicken BEST1-Fab complexes, at 2.85 Å resolution, with permeant anions and Ca²⁺. The eukaryotic BEST1 channel recapitulates CaCC function in liposomes and is formed from a pentameric assembly of subunits. Ca²⁺ binds to the channel's large cytosolic region. A single ion pore, approximately 95 Å in length, is located along the central axis and contains at least 15 binding sites for anions. A hydrophobic neck within the pore probably forms the gate. Phenylalanine residues within it may coordinate permeating anions via anion-π interactions. Conformational changes observed near the 'Ca²⁺ clasp' hint at the mechanism of Ca²⁺-dependent gating. Disease-causing mutations are prevalent within the gating apparatus (Dickson et al. 2014).

The bestrophin family of calcium (Ca²⁺)-activated chloride (Cl^-) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca²⁺, comprises four members in mammals (bestrophin 1-4). Owji et al. 2020 reported cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca²⁺ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis revealed that, although the channel is sensitive to Ca²⁺, it has substantial Ca²⁺-independent activity for Cl^-, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway, even when Ca²⁺ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating (Owji et al. 2020).

Many transmembrane proteins are modulated by intracellular or extracellular pH. Investigation of pH dependence generally proceeds by mutagenesis of a wide set of amino acids, guided by properties such as amino-acid conservation and structure. Prediction of pKas can streamline this process, allowing rapid and effective identification of amino acids of interest with respect to pH dependence. Commencing with the calcium-activated chloride channel, bestrophin 1, the carboxylate ligand structure around calcium sites relaxes in the absence of calcium, consistent with a measured lack of pH dependence (Elverson et al. 2023). By contrast, less relaxation in the absence of calcium in TMEM16A, and maintenance of elevated carboxylate sidechain pKas, is suggested to give rise to pH-dependent chloride channel activity. This hypothesis, modulation of calcium/proton coupling and pH-dependent activity through the extent of structural relaxation, applies to the well-characterised cytosolic proteins calmodulin (pH-independent) and calbindin D9k (pH-dependent). Further application of destabilized, ionisable charge sites, or electrostatic frustration, is made to other human chloride channels (that are not calcium-activated), ClC-2, GABA(A), and GlyR. Experimentally determined sites of pH modulation have been identified. Structure-based tools for pKa prediction are available, allowing users to focus on mutagenesis studies, construct hypothetical proton pathways, and derive hypotheses such as the model for control of pH-dependent calcium activation through structural flexibility (Elverson et al. 2023). 

The reaction catalyzed by functionally characterized members of the Bestrophin family is:

Anions (out) ⇌ anions (in)