Abstract
The voltage-dependent anion channel (VDAC) governs the free exchange of ions and metabolites between the mitochondria and the rest of the cell. The three-dimensional structure of VDAC1 reveals a channel formed by 19 β-strands and an N-terminal α-helix located near the midpoint of the pore. The position of this α-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The participation of the N-terminus of VDAC1 in the voltage-gating process has been well established, but the molecular mechanism continues to be debated; however, the majority of models entail large conformational changes of this N-terminal segment. Here we report that the pore-lining N-terminal α-helix does not undergo independent structural rearrangements during channel gating. We engineered a double Cys mutant in murine VDAC1 that cross-links the α-helix to the wall of the β-barrel pore and reconstituted the modified protein into planar lipid bilayers. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with β-strand 11 of the pore wall during voltage gating.
Highlights
There is ongoing controversy concerning the location and mobility of the N-terminal ␣-helix in VDAC1 during voltage gating
There is considerable evidence demonstrating that voltage-dependent anion channel (VDAC) undergoes large conformational changes during voltage-dependent gating [1, 13, 14], and many studies have attributed these changes to movements of the N-terminal segment [7, 17, 18, 20, 23, 32, 37]
By a combination of site-directed mutagenesis, induced disulfide bond formation, and electrophysiological characterization in planar lipid bilayers, we have shown that VDAC with the N-terminal ␣-helix portion engineered to covalently bind the pore wall behaves indistinguishably from endogenous protein, suggesting that the N-terminal segment is either not mobile or does not move independently of the rest of the voltage-sensor domain during gating
Summary
There is ongoing controversy concerning the location and mobility of the N-terminal ␣-helix in VDAC1 during voltage gating. Results: mVDAC1 with the N-terminal ␣-helix cross-linked to -strand 11 forms typical voltage-gated channels. The three-dimensional structure of VDAC1 reveals a channel formed by 19 -strands and an N-terminal ␣-helix located near the midpoint of the pore. The position of this ␣-helix causes a narrowing of the cavity, but ample space for metabolite passage remains. The modified murine VDAC1 exhibited typical voltage gating. These results suggest that the N-terminal ␣-helix is located inside the pore of VDAC in the open state and remains associated with -strand 11 of the pore wall during voltage gating
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