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. 2011 Apr 29;6(4):e19364.
doi: 10.1371/journal.pone.0019364.

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus

Vladimir M Milenkovic  1 , Sarka Krejcova, Nadine Reichhart, Andrea Wagner, Olaf Strauss
Affiliations

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus

Vladimir M Milenkovic et al. PLoS One. .

Abstract

Bestrophin-1 modulates currents through voltage-dependent L-type Ca(2+) channels by physically interacting with the β-subunits of Ca(2+) channels. The main function of β-subunits is to regulate the number of pore-forming Ca(V)-subunits in the cell membrane and modulate Ca(2+) channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca(2+) channel subunits, together with modulation of Ca(V)1.3 Ca(2+) channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and Ca(V)1.3 subunits partly lost membrane localization. Currents from Ca(V)1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca(2+) channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming Ca(V) Ca(2+)-channel subunits.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Interaction between pore-forming CaV1.3 and auxiliary β-subunits of voltage-dependent Ca2+ channels.
1A: Left panel: CHO cells were transfected with CaV1.3-GFP fusion construct and β3-subunits, precipitated with antibodies against β3-subunits and blotted for CaV1.3 protein. Proteins precipitated using antibodies against GFP were positively stained with antibodies against CaV1.3 indicating identification of CaV1.3 subunits. Right two panels: CHO cells transfected with CaV1.3 and β3-subunits; CaV1.3 was immunoprecipitated and blotted for β3-subunits. Control experiment: precipitation using anti-β3-antibody and blot stained against β3-subunit. (L  =  lysate, 10% of total protein; IP  =  immunoprecipitation) 1B: ARPE-19 cells transfected with β3-subunit (green) and CaV1.3 (red). The merged picture shows co-localization of β3-subunits and CaV1.3. On the right: fluorescence profile showing subcellular protein distribution. 1C: To quantify plasma membrane localization, pixel analysis was performed for edge detection to calculate surface expression (data are mean ± SEM; n = 3). Scale bar represents 10 µm.
Figure 2
Figure 2. Subcellular localization of heterologously expressed CaV1.3, β-subunits of voltage-dependent Ca2+ channels and bestrophin-1:
ARPE-19 cells were transfected with: β3-subunits and bestrophin-1, CaV1.3, β3 or β4-subunits and bestrophin-1, and human P2Y2-His6 receptor. 2A: Cells transfected with β3-subunit (green) and bestrophin-1 (red). Yellow colour in the merged picture indicates interaction of both proteins. On the right: fluorescence profiles showing subcellular protein distribution. 2B: Cells transfected with β3-subunit (green), bestrophin-1 (red), and Cav1.3 subunit (blue). White colour in the merged picture suggests co-localization of all three proteins. On the right: fluorescence profiles showing subcellular protein distribution. 2C: Cells transfected with β4-subunit (green), bestrophin-1 (red), and Cav1.3 subunit (blue). White colour in the merged picture suggests co-localization of all three proteins. On the right: fluorescence profiles showing subcellular protein distribution. 2D: Human P2Y2-His6 receptor which shows plasma membrane localization as a control. Note: cells which express CaV1.3 and β-subunits always appear in a more spherical shape and do not remain flat due to the expression of the large L-type channel subunits. The smaller P2Y2-receptor did not change the cell shape. 2E: Relative surface expression quantified by edge detection analysis (data are mean ± SEM; n = 3). (*  =  p<0.05 for bestrophin-1; #  =  p<0.05 for β3-subunits, unpaired t-test) Scale bar represents 10 µm.
Figure 3
Figure 3. Detection of interaction sites between β-subunits and bestrophin-1.
3A: Bestrophin-1 construct used in this study and alignment of amino acid sequences of the C-terminus of the bestrophin-1 from different species (Boxes: transmembrane domains). Two among vertebrate species highly conserved clusters of proline-rich motifs (PxxP) could be detected. In the ΔCTPxxP mutant form, PxxP motifs between amino acid 468 to 486 were removed with unchanged recognition sites for the anti bestrophin-1 antibody. 3B: HEK-293 cells were transfected with β3-subunits together with bestrophin-1 or ΔCTPxxP constructs. Proteins were precipitated using anti-bestrophin-1 antibody and blots were visualized for anti-β3-subunit to show co-immunoprecipitation. 3C: HEK-293 cells were transfected with His-tagged β4-subunits together with bestrophin-1 wild type or ΔCTPxxP constructs. Proteins were precipitated using anti-His antibody and the blots were visualized with anti-bestrophin-1 antibody to show co-immunoprecipitation. 3D: Relative co-immunoprecipitation of β3-subunits with either wild-type or ΔCTPxxP bestrophin-1: efficiency was measured by densitometry (n = 5). 3E: Relative co-immunoprecipitation of β4-subunits with either wild-type or ΔCTPxxP bestrophin-1 (depicted n = 3). (L  =  lysate; IP  =  immunoprecipitation; NB  =  not bound). The following species abbreviations were used: Hs, Homo sapiens, Mm, Macaca mulatta, Bt, Bos taurus, Ms, Mus musculus, Xt, Xenopus tropicalis, Fr, Fugu rubripes, and Ci, Ciona intestinalis.
Figure 4
Figure 4. Complex formation of CaV1.3, β4-subunits and bestrophin-1.
HEK cells were transfected with CaV1.3, His-tagged β4-subunits, bestrophin-1 or ΔCTPxxP bestrophin-1. Immunoprecipitation was performed using anti-CaV1.3 antibodies, precipitates were analyzed by Western blot. 4A: Transfection: CaV1.3 and bestrophin-1. Blot staining with anti-CaV1.3 antibody to show efficient precipitation of CaV1.3 subunits. 4B: Transfection: CaV1.3 and bestrophin-1. Blot staining with anti-bestrophin-1 antibody showing no co-precipitation of CaV1.3 subunits with bestrophin-1. 4C: Transfection: CaV1.3 and ΔCTPxxP bestrophin-1. Blot staining with anti-bestrophin-1 antibody showing no co-precipitation of CaV1.3 subunits with ΔCTPxxP bestrophin-1. 4D: Transfection: CaV1.3, His-tagged β4-subunits and bestrophin-1. Blot staining with anti-His antibody showing co-precipitation of CaV1.3 subunits with β4-subunits. 4E:Transfection: CaV1.3, His-tagged β4-subunits and bestrophin-1. Blot staining with anti-bestrophin-1 antibody showing indirect co-precipitation of CaV1.3 subunits with bestrophin-1. 4F: Transfection: CaV1.3, His-tagged β4-subunits and ΔCTPxxP bestrophin-1. Blot staining with anti-His antibody showing co-precipitation of CaV1.3 subunits with β4-subunits. 4G: Transfection: CaV1.3, His-tagged β4-subunits and ΔCTPxxP bestrophin-1. Blot staining with anti-bestrophin-1 showing indirect co-precipitation of CaV1.3 subunits with ΔCTPxxP bestrophin-1. (L  =  lysate; IP  =  immunoprecipitation; NB  =  not bound)
Figure 5
Figure 5. Patch-Clamp analysis of CaV1.3/β4 currents under influence of bestrophin-1.
5A: Whole cell Ba2+-currents (lower panel) measured from CHO cells expressing CaV1.3, β4-, α2δ1-subunits. Currents were elicited by a series of 9 voltage-steps of 10 mV increasing amplitude and 50 ms duration from a holding potential of -70 mV (upper panel). 5B: Gating currents: Currents elicited by the electrical stimulation as shown in 5A in the same cell after switching to Co2+ containing bath solution. 5C: Plot of normalized gating currents to the membrane voltages of the electrical stimulation (mean ± SEM, n = 3). 5D: Comparison of the ionic current density of L-type currents from heterologously expressed Ca2+ channel proteins and different bestrophins measured at +20 mV (note: the values are close to those we have published but represent a different set of data). 5E: Comparison of the gating current density in the presence of 10 mM Co2+ from heterologously expressed Ca2+ channel proteins and different bestrophins measured at +20 mV. 5F: Normalized current/voltage plots of L-Type currents either in the absence, presence of bestrophin-1 or the ΔCTPxxP mutant bestrophin-1, fit by Boltzmann equation. 5G: Comparison of the voltages of half maximal activation obtained from Boltzmann fits of the curves in Fig. 5F. 5H: Whole cell Ba2+-currents measured from CHO cells expressing either CaV1.3, β4-, α2δ1 subunits or CaV1.3, β4-, α2δ1 subunits plus wt-bestrophin-1. Currents were elicited by a voltage-jump from -70 mV to +20 mV. The recording shows the currents normalized to their maximal amplitude for comparison. 5I: Comparison of the time-dependent activation of L-type currents from different heterologously expressed Ca2+ channel proteins and bestrophins measured as time constant from single-exponential fit of the currents (note: the values are close to those we have published in but represent a different set of data). (*  =  p<0.05; **  =  p<0.01, unpaired t-test; n depicts the number of experiments)
Figure 6
Figure 6. Subcellular localization of Ca2+ channel subunits and bestrophin-1 in cells used for patch-clamp analysis.
6A: Confocal pictures of CHO cells expressing wt-bestrophin-1, β4-subunit, α2δ1-subunit and CaV1.3. The merged picture and the fluorescence profiles measured along the red line (right panel) indicate the presence of β4-subunit, CaV1.3 and wt-bestrophin-1 in the cell membrane. 6B: Confocal pictures of CHO cells expressing ΔCTPxxP bestrophin-1, β4-subunit, α2δ1-subunit and CaV1.3. The merged picture and the fluorescence profiles measured along the red line (right panel) indicate the presence of β4-subunit, CaV1.3 and ΔCTPxxP bestrophin-1 in the cell plasma. 6C: Confocal pictures of CHO cells expressing ΔCTPxxP bestrophin-1 and P2Y2 receptor. The merged picture and the fluorescence profiles measured along the red line (right panel) indicate the presence of ΔCTPxxP bestrophin-1 in the cell plasma whereas the P2Y receptor appears in the cell membrane. 6D: Relative surface expression quantified by edge detection analysis (data are mean ± SEM; n = 3). (*  =  p<0.05; **  =  p<0.01, unpaired t-test) Scale bar represents 10 µm.
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