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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).

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.

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

Anions (out) ⇌ anions (in)

References associated with 1.A.46 family:

Bharill, S., Z. Fu, R. Palty, and E.Y. Isacoff. (2014). Stoichiometry and specific assembly of Best ion channels. Proc. Natl. Acad. Sci. USA 111: 6491-6496. 24748110
Burgess, R., I.D. Millar, B.P. Leroy, J.E. Urquhart, I.M. Fearon, E. De Baere, P.D. Brown, A.G. Robson, G.A. Wright, P. Kestelyn, G.E. Holder, A.R. Webster, F.D. Manson, and G.C. Black. (2008). Biallelic mutation of BEST1 causes a distinct retinopathy in humans. Am. J. Hum. Genet. 82: 19-31. 18179881
Carter, D.A., M.J. Smart, W.V. Letton, C.M. Ramsden, B. Nommiste, L.L. Chen, K. Fynes, M.N. Muthiah, P. Goh, A. Lane, M.B. Powner, A.R. Webster, L. da Cruz, A.T. Moore, P.J. Coffey, and A.F. Carr. (2016). Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC). Sci Rep 6: 33792. 27653836
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Hagen, A.R., R.D. Barabote, and M.H. Saier. (2005). The bestrophin family of anion channels: identification of prokaryotic homologues. Mol. Membr. Biol. 22: 291-302. 16154901
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Kane Dickson, V., L. Pedi, and S.B. Long. (2014). Structure and insights into the function of a Ca2+-activated Cl- channel. Nature 516: 213-218. 25337878
Mladenova, K., S.D. Petrova, T.D. Andreeva, V. Moskova-Doumanova, T. Topouzova-Hristova, Y. Kalvachev, K. Balashev, S.S. Bhattacharya, C. Chakarova, Z. Lalchev, and J.A. Doumanov. (2016). Effects of Ca2+ ions on bestrophin-1 surface films. Colloids Surf B Biointerfaces 149: 226-232. [Epub: Ahead of Print] 27768912
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Qu, Z. and H.C. Hartzell. (2008). Bestrophin Cl- channels are highly permeable to HCO3-. Am. J. Physiol. Cell Physiol. 294: C1371-1377. 18400985
Qu, Z., W. Cheng, Y. Cui, Y. Cui, and J. Zheng. (2009). Human disease-causing mutations disrupt an N-C-terminal interaction and channel function of bestrophin 1. J. Biol. Chem. 284: 16473-16481. 19372599
Qu, Z., Y. Cui, and C. Hartzell. (2006). A short motif in the C-terminus of mouse bestrophin 3 [corrected] inhibits its activation as a Cl channel. FEBS Lett. 580: 2141-2146. 16563389
Roberts, S.K., J. Milnes, and M. Caddick. (2011). Characterisation of AnBEST1, a functional anion channel in the plasma membrane of the filamentous fungus, Aspergillus nidulans. Fungal Genet Biol 48: 928-938. 21596151
Stotz, S.C. and D.E. Clapham. (2012). Anion-sensitive fluorophore identifies the Drosophila swell-activated chloride channel in a genome-wide RNA interference screen. PLoS One 7: e46865. 23056495
Sun, H., T. Tsunenari, K.-W. Yau, and J. Nathans. (2002). The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc. Natl. Acad. Sci. USA 99: 4008-4013. 11904445
Tsunenari, T., H. Sun, J. Williams, H. Cahill, P. Smallwood, K.-W. Yau, and J. Nathans. (2003). Structure-function analysis of the Bestrophin family of anion channels. J. Biol. Chem. 278: 41114-41125. 12907679
Woo, D.H., K.S. Han, J.W. Shim, B.E. Yoon, E. Kim, J.Y. Bae, S.J. Oh, E.M. Hwang, A.D. Marmorstein, Y.C. Bae, J.Y. Park, and C.J. Lee. (2012). TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation. Cell 151: 25-40. 23021213
Xiao, Q., A. Prussia, K. Yu, Y.Y. Cui, and H.C. Hartzell. (2008). Regulation of bestrophin Cl channels by calcium: role of the C terminus. J Gen Physiol 132: 681-692. 19029375
Xiao, Q., K. Yu, Y.Y. Cui, and H.C. Hartzell. (2009). Dysregulation of human bestrophin-1 by ceramide-induced dephosphorylation. J. Physiol. 587: 4379-4391. 19635817
Yang, T., Q. Liu, B. Kloss, R. Bruni, R.C. Kalathur, Y. Guo, E. Kloppmann, B. Rost, H.M. Colecraft, and W.A. Hendrickson. (2014). Structure and selectivity in bestrophin ion channels. Science 346: 355-359. 25324390
Yu, K., Q. Xiao, G. Cui, A. Lee, and H.C. Hartzell. (2008). The best disease-linked Cl- channel hBest1 regulates Ca V 1 (L-type) Ca2+ channels via src-homology-binding domains. J. Neurosci. 28: 5660-5670. 18509027
Yu, K., Y. Cui, and H.C. Hartzell. (2006). The bestrophin mutation A243V, linked to adult-onset vitelliform macular dystrophy, impairs its chloride channel function. Invest Ophthalmol Vis Sci 47: 4956-4961. 17065513