Effects of alternative splicing on the function of bestrophin-1 calcium-activated chloride channels

Abstract

The proposed Ca2+-activated Cl- channel protein Best1 (bestrophin 1) is expressed and functionally important in the retina and in the brain. Human BEST1 has two known splice variants, Best1V1 and Best1V2, which arise from alternative splicing of two exons: exon 2 splicing results in a unique N-terminal domain, whereas alternative splicing of exon 11 produces two mutually exclusive C-termini. Prior studies were limited to Best1V1 and its clinically relevant mutations. In the present work, we cloned a novel splice variant of Best1V1 missing exon 2 (Best1V1Δex2) and differing from each of the two previously identified isoforms by one alternatively spliced domain. This finding allowed us to determine the role for alternative splicing of the Best1 N- and C-termini. We heteroexpressed Best1V1Δex2 in HEK (human embryonic kidney)-293 cells, and compared its properties with Best1V1 and Best1V2. Western blot analysis confirmed protein expression from all three splice variants. Both Best1V1 and Best1V1Δex2 successfully formed Ca2+-activated Cl- channels, demonstrating that the N-terminus encoded by exon 2 is not essential for channel function. In contrast, Best1V2-expressing cells had no detectable Ca2+-activated Cl- currents, pointing to a critical role for splicing of the C-terminus. Surface protein biotinylation demonstrated that Best1V1 and Best1V1Δex2 are trafficked to the plasma membrane, whereas Best1V2 is not. These results define the impact of alternative splicing on Best1 function, and should be taken into consideration in future modelling of the Best1 protein structure.

Figure 1. Schematic representation of the Bestrophin-1 gene splice variants (A) and their predicted protein products (B)

A, The Best1 gene consists of 11 exons. The Best1V1 splice variant (NM_004183) is composed of all 11 exons, with initiation of translation (arrow) in exon 2 and termination of translation (asterisk) in exon 11. In Best1V2 (NM_001139443), exon 2 (solid black) is removed due to alternative splicing, and translation is initiated from a start codon in exon 3. Additionally, Best1V2 does not splice out the intron between exons 10 and 11, and the C-terminus is encoded by translation into the intronic sequence (open box with doted border). The newly cloned Best1V1Δex2 (GenBank accession number JQ954696), is similar to Best1V1 except that it is missing exon 2. B, The Best1 protein model proposed by Milenkovic et al. [16]. This model depicts the predicted membrane topology of canonical Best1V1. In Best1V1Δex2, the first 60 amino N-terminal acids (solid black) are missing. In Best1V2, the first 60 N-terminal amino acids (black) are missing, and the last five amino acids on C-terminus (multipoint stars) are replaced with unique 84 amino-acid sequence.

Figure 2. Western blot analysis of previously cloned splice variants of Best1 heteroexpressed in HEK293 cells

A, Best1V1 and Best1V2 were heteroexpressed in HEK293 cells and probed with a monoclonal anti-Best1V1 antibody. B, Best1V1, Best1V2, and Best1V2 with a DDK epitope fused to the C-terminus (Best1V2-DDK) were heteroexpressed in HEK293 and probed with a polyclonal antibody recognizing Best1V1 and Best1V2. C, The same cell lysates as in B were probed with an anti-DDK antibody in order to assure specificity of the Best1V2 signal. For all Western blots the membranes were stripped and reprobed with a monoclonal anti-actin antibody to assess equality of loading (shown in boxes below). The figures are representative images of at least three Western blots performed in three independent cell transfections. Abbreviations: MW, molecular weight standards; WT, lysates prepared from mock-transfected (wild type) cells; Act, actin immunoreactivity. IR = immunoreactivity.

Figure 3. Electrophysiological characterization of canonical splice variants of Best1 heteroexpressed in HEK293 cells

A-C, Representative traces of chloride currents in response to step pulses from -100 mV to +100 mV in 20 mV increments in non-transfected (Wild Type) cells and HEK cells with heterologous expression of Best1V1 or Best1V2. D-E, Representative traces showing the current-voltage relationship of heteroexpressed Best1V1 channels in the absence and presence of 200 μM niflumic acid (NFA). F, The I-V plot demonstrating effect of niflumic acid on Cl⁻ currents in Best1V1-expressing cells. G-I, Summary of the average Cl⁻ current densities in non-transfected HEK cells (G) and HEK cells heterologously expressing Best1V1 (H), or Best1V2 (I). Data are the mean values ±SE of average Cl⁻ current densities recorded from at least two independent cell transfections, with the total number of cells indicated in parentheses. Recordings were performed with [Ca²⁺] in the pipette solution buffered at 0 or 1 μM, in the presence or absence of 200 μM NFA. **p<0.01, vs. currents recorded at [Ca²⁺]_p=0 μM; ^#p<0.05 vs. currents recorded in the absence of NFA.

Figure 4. Functional characterization of the novel Best1V1 splice variant missing exon 2 by heterologous expression in HEK293 cells

A, Western blot analysis of Best1V1 and Best1V1Δex2 heteroexpressed in HEK293 and probed with a monoclonal anti-Best1 antibody. Untransfected HEK293 (WT) cell lysates shown as a control for signal specificity. The Western blots membranes were stripped and reprobed with a monoclonal anti-actin antibody to compare protein loading (shown in box below). The figure is a representative image of three Western blots in independent cell transfections. B, Representative traces of Best1V1Δex2-mediated Cl⁻ currents recorded in response to step pulses from −100 mV to +100 mV in 20 mV increments. C, Representative traces showing the current-voltage relationship of heteroexpressed Best1V1Δex2 channels in the absence and presence of 200 μM niflumic acid (NFA). D, Summary of the electrophysiological recording performed in HEK293 cells expressing Best1V1Δex2. Data are the mean values ±SE of average current densities recorded with [Ca²⁺] in the pipette solution buffered at 0 or 1 μM, in the presence or absence of 200 μM NFA. The recordings were done in two independent cell transfections, with the number of cells indicated above each bar. Not all Best1V1Δex2-trensfectected cells showed Ca²⁺-sensitive Cl⁻ currents (see text for further details). ***p<0.001, vs. currents recorded at [Ca²⁺]_pipette = 0 μM; ^##p<0.01 vs. currents recorded in the absence of NFA.

Figure 5. Biotinylation assay of the surface protein expression for three distinct splice variants of Best1 expressed in HEK293 cells

A, Western blot analysis of precipitated biotinylated fraction and the crude cell lysates in HEK293 cells expressing the DDK-tagged Best1V1. Representative of four blots. Because of the difference in the expression levels/intensity of the signal, Best1V1-DDK has been probed separately from Best1V1Δex2-DDK and Best1V2. B, Western blot analysis of biotinylated (surface) material and the crude cell lysates in HEK293 expressing Best1V1Δex2-DDK and Best1V2-DDK. Representative of three blots. Asterisks in (B) indicate two very weak immunoreactive bands in cells expressing Best1V2-DDK. Boxes below each panel show actin immunoreactivity on the same membrane after stripping. These controls show lack of cytosolic proteins in biotinylated fraction.