Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking

Abstract

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

“…4 B, C, and G). In addition, coexpression of AQP1/hBEST1 and BEST1-A243V also leads to significant reduction in VRAC currents, in agreement with a dominant-negative mode of action of BEST1 mutations on wild-type BEST1, as suggested earlier (37).…”

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a NovelBEST1Mutation

“…6 Monomers of Best1 have four transmembrane domains as well as intracellular N-and C-termini, with the latter being at the terminus of a large, cytosolic domain. [7][8][9] Monomers of Best1 oligomerize [10][11][12][13][14] to form a highly conserved homopentameric Ca 2þ -activated ion channel. 8,9 Data from heterologous systems, 15,16 primary human RPE cell culture, 17,18 mouse models, 19,20 the structurally similar paralog bestrophin-2, 21,22 as well as recently published crystal structures 8,9 all unambiguously demonstrate that mammalian Best1 is a calcium-activated anion channel.…”

“…We maintained HEK293 and MDCK II cells (American Type Culture Collection, Manassas, VA, USA) as previously described 10,11 : in a 95% air 5% CO 2 environment at 378C. Transfections were carried out as before 10,11,17 using a commercial reagent (Lipofectamine; Invitrogen, Carlsbad, CA, USA).…”

“…10,11 We synthesized the cDNA for the novel Best1 mutant synthetically (Integrated DNA Technologies, Coralville, IA, USA) and spliced into the NheI and AgeI restriction sites of pECFP-N1 (Clontech Laboratories, Inc., Mountain View, CA, USA) in-frame with CFP. To generate a replication-defective adenovirus encoding for this mutant fused to CFP, Best1…”

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

“…As the stoichiometry was unknown (Johnson et al, 2013) but was expected to be between four and six subunits per channel (with the mass of a channel-Fab complex therefore being of the order of 300-500 kDa), it was concluded that perhaps the difficulty in phasing with bromide or selenium was owing to the large number of relatively weak sites produced by these derivatives, and phasing may be made possible by using a large cluster compound that could bind at a single site per subunit or even per channel. The only heavy-atom cluster tested for this protein crystal was tantalum bromide (Ta 6 Br 12 ÁBr 2 ), but several others are now commercially available, including the magic triangle (Beck et al, 2009) and tungsten-cluster salts (Rudenko et al, 2003), that may be useful in similar circumstances by the same rationale.…”