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Figure 6
Figure 6. E73 is the critical amino acid residue that determines the ability of VDAC2 to suppress cardiac fibrillation in the tremblor/ncx1h mutant. (A) Alignment of protein sequences of VDAC1, 2, and 3 from different species. In all the species examined the position corresponding to zebrafish residue 73 is consistently occupied by a glutamate (E) in VDAC1 and VDAC2, whereas this position is occupied by glutamine (Q) in VDAC3. (B) Three-dimensional model of the VDAC2 protein (pdb: 4bum) showing the location of amino acid 73 in β-sheet 4 in red. (C) Western blot analysis of lysates from 30 hpf uninjected embryos or embryos injected with 25 pg. FLAG-tagged wild type and point mutant VDAC mRNA. β-actin was used as a loading control. (D) Mutation of E73 to Q in VDAC2 abrogates its ability to suppress cardiac fibrillation in tremblor/ncx1h mutants (49.7 ± 2.8%, N = 3, n = 144 with VDAC2 in contrast to 21.7 ± 5.1%, N = 3, n = 155 with VDAC2E73Q). (E) Vice-versa, by mutating Q73 to E, VDAC3 gained the ability to restore rhythmic cardiac contraction in tre mutants (19.0 ± 4.4%, N = 3, n = 182 with VDAC3 in contrast to 47.2 ± 4.3%, N = 3, n = 145 with VDAC3Q73E). Overall rescue percentages represent the mean rescue percentage ± s.e.m. from N independent experiments, using a total of n embryos.