The Human Mitochondrial DNA Depletion Syndrome Gene MPV17 Encodes a Non-selective Channel That Modulates Membrane Potential

Abstract

The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.

Keywords: Mpv17 protein; aging; membrane protein; mitochondria; mitochondrial DNA damage; mitophagy; non-selective channel.

FIGURE 1.

In silico analysis of MPV17 sequence. A, a phylogenic tree was constructed using amino acid sequences of human (PXMP2, MPV17, MP-L, and FKSG24/MPV17L2) and yeast Saccharomyces cerevisiae (Sym1 and Yor292) proteins that constitute the Pxmp2 family (see also Ref. 14). B, sequence of human MPV17 protein. Charged amino acids are colored as follows: Asp and Glu (red); Arg and Lys (blue); and His (green). The putative membrane-spanning domains predicted using the HMMTOP algorithm are shown in boxes. For the detection of putative α-helices, four different programs were used (see “Experimental Procedures” for details). The α-helices predicted by at least three of four applied programs and long enough to penetrate the membrane lipid bilayer (18 amino acids) were chosen for further analysis (marked in boldface italic type and underlined). Note that the putative membrane-spanning domains that are supposed to be α-helices (shown in boxes) do not necessarily coincide with the predicted α-helices (marked in boldface italic type). The locations of point mutations leading to MDDS are marked by asterisks. The aspartic acid (D) determining ion selectivity of the channel is marked by red box. The putative phosphorylation sites are shown by blue boxes. C, helical wheel representations of the sequences underlined in B. Amino acids are colored according to the physico-chemical properties of the side chains: hydrophobic (yellow); polar, uncharged (green); negatively charged (pink); and positively charged (blue). The sequences of the corresponding α-helices are shown over the wheels. Dotted lines separate mainly hydrophilic sides from mostly hydrophobic regions of the sequences.

FIGURE 2.

Isolation of recombinant MPV17 protein and detection of channel-forming activity. A, size exclusion chromatography of MPV17 after His tag affinity purification step. Positions of molecular mass markers are shown. B, Coommassie-stained SDS-PAGE of MPV17 samples obtained after His trap (far left lane) and size exclusion (other lanes) chromatography. The position of MPV17 is marked by an asterisk. C, secondary structure analysis of purified MPV17 using CD spectroscopy. D, MCR of isolated MPV17. Top, current trace of a membrane bilayer showing the insertion of multiple channels. Electrolyte was symmetrical 1.0 m KCl buffered with 10 mm Tris-Cl, pH 7.2. Bottom, histogram of the frequency of insertion events relative to their conductance. Bin size was 2 pA. The total number of calculated insertion events (I.e.) is indicated. E, long term recording of isolated MPV17 channel (see the legend to Fig. 2D for details). Note that the channels are permanently open for several min. F, single channel conductance of purified MPV17 as a function of KCl concentration (n = 6). Error bars, S.D.

FIGURE 3.

SCA of isolated MPV17 protein. A, recording of a single channel current using the indicated voltage-step protocol. Electrolyte (A, B, C, E, and G) was symmetrical 1.0 m KCl buffered with 10 mm Tris-Cl, pH 7.2. B, activity of a single channel was registered at moderate membrane potentials (±60 mV) and long term resolution. C, current-voltage relationship of a single MPV17 channel. D, current-voltage dependence of a single channel under asymmetric electrolyte conditions: 1.0 m KCl trans/0.5 m KCl cis (1) or 0.5 m CaCl₂ trans/0.15 M CaCl₂ cis (2). E, current trace of a single channel in response to the indicated voltage-ramping. F, voltage-dependent open probability (P_open) of the MPV17 channel. G, application of the polymer-exclusion method to estimate the size of the channel's pore. Left, 30–40 single insertion events registered without (G₀) or with (G) non-electrolyte by SCA were used to calculate the average conductance of the channel. The data are presented as ratio G/G₀ plotted against hydrated radii of non-electrolytes (see “Experimental Procedures” for details). Right, second derivative of the dependence from the left panel, indicating the maximal and the minimal turning points. The upper point (0.7 nm) predicts the molecular radius of solutes at which movement inside the pore becomes restricted. The lower point (0.9 nm) indicates the minimal radius of molecules that are not conducted by the channel. Error bars, S.D.

FIGURE 4.

Recordings of a single MPV17 channel at the different membrane potentials indicated. The protein was isolated without DTT. A, current recording of a channel (insertion event is marked by a single asterisk) at successive step increases of positive and negative potentials. Electrolyte (A, C, and E) was symmetrical 1.0 m KCl (without DTT) buffered with 10 mm Tris-Cl, pH 7.2. Time scale-magnified traces of the insertion event (left) and channel closing (below panels) are shown. B, amplitude histogram derived from recording of the channel activity at −120 mV shown in A (the region of analysis is marked by a heavy line and two asterisks). Note several subconductance states of the channel, which are numbered starting from the fully open state. C, closing of a single MPV17 channel at +150 mV. A time scale-expanded trace is shown at the bottom. D, amplitude histogram of the recording shown in C. Note the similarity in the amplitudes derived at negative (B) and positive (D) membrane potentials and showing four subconductance states, where state 1 comprises two substates. E, insertion of a single channel (MCR) in a fully open conformation, indicating fluctuations of the current that apparently reflect transitions between two substates of the subconductance state 1 (see B and D for a comparison).

FIGURE 5.

Redox sensitivity of the purified MPV17 channel; effect of H₂O₂ and DTT. A, MPV17 was isolated using a standard protocol and treated with 200 μm H₂O₂ for 30 min, and the peroxide was removed by dialysis. Shown is a current trace of a single channel in response to the indicated voltage ramp protocol. Electrolyte (A–E) was symmetrical 1.0 m KCl, pH 7.2. Note that H₂O₂ treatment did not affect conductance of the channel. The bottom trace represents time scale-expanded recording of the top trace and shows fluctuations of current amplitudes at the fully open state of the channel, which correspond to subconductance state 1 in Fig. 4, B and D. These fluctuations are characteristic for the channels pretreated with H₂O₂ and may cause some variations in conductance values obtained for the MPV17 channel at different conditions. B, current traces at a long term resolution indicating that at oxidative conditions (see above), the channel is in an open conformation even at elevated membrane potentials (±100 mV). C, voltage-dependent open probability (P_open) of MPV17 channel pretreated with 200 μm H₂O₂ (1) or 10 mm DTT (2) or isolated in the presence of 5 mm DTT (3); the measurements were made without (1) or with (2 and 3) 10 mm DTT (n = 6–8). D, current-voltage dependence of a single MPV17 channel preincubated with 10 mm DTT. Electrolyte (D and E) contained 10 mm DTT. The bottom traces represent time scale-expanded current recording of the top trace, indicating closing of the channel. E, current trace of two channels in response to the shown voltage ramping. MPV17 protein was isolated in the presence of 5 mm DTT. The bottom traces represent time scale-expanded recordings of the top trace, showing an appearance of subconductance states during closing of the channels. Error bars, S.D.

FIGURE 6.

Effect of pH on MPV17 channel activity when pretreated with DTT. Electrolyte was symmetrical 1.0 m KCl containing 10 mm DTT and buffered with 10 mm Tris-Cl, pH 7.2 (A) and pH 8.2 (B and C), or with 10 mm sodium acetate, pH 5.8 (D). A, closing of a single channel at step increase of membrane potential to +80 mV (top). Bottom, amplitude histogram derived from the recording shown at the top. Note that the only two subconductance states were registered. B, insertion of a single channel at pH 8.2. C, current trace of a channel shown in B in response to the indicated voltage ramp protocol. The bottom trace represents a time scale-expanded recording of the top trace. All four subconductance states (numbered) were registered. D, current-voltage dependence of a single channel registered at pH 5.8. Note the partial closing of the channel at high membrane potential. E, voltage-dependent open probability (P_open) of the channel at pH 5.8 (1) or pH 8.2 (2) (n = 4–6). Electrolyte was symmetrical 1.0 m KCl containing 10 mm DTT and buffered with 10 mm sodium acetate (1) or 10 mm Tris-Cl (2). Error bars, S.D.

FIGURE 7.

SCA of MPV17 protein with mutation p.D92K. A, current trace of a single channel. Electrolyte (A, B, and D) was symmetrical 1.0 m KCl, pH 7.2. B, current-voltage relationship of a mutant channel (electrolyte contained 10 mm DTT) (n = 8–10). C, current-voltage relationship of the channel under asymmetric electrolyte conditions: 1.0 m KCl trans/0.5 m KCl cis (1) or 0.5 m CaCl₂ trans/0.15 m CaCl₂ cis (2); all samples contained 10 mm DTT (n = 8–10). D, current trace of the channel in response to the indicated voltage ramp protocol. The medium was without (top) or with (bottom) 10 mm DTT. E, voltage-dependent open probability (P_open) of the mutant channel in presence of 10 mm DTT; n = 4–6. Error bars, S.D.

FIGURE 8.

Electrophysiological analyses of MPV17 protein carrying mutations p.T80D or p.T80A. A, MCR of the p.T80D mutant channel. The low conductance insertions are marked by asterisks. Electrolyte (A, C, and D–F) was symmetrical 1.0 m KCl, pH 7.2, containing no DTT. B, histogram of the frequency of insertion events relative to their conductance. Bin size was 2 pA. Top, p.T80D channel; bottom, p.T80A channel. C, SCA recording of insertion of the two p.T80D channels. The low conductance insertion is marked by an asterisk. D, closing of a single p.T80D channel at positive voltages. The bottom trace represents time scale-expanded recording of the top trace. Note that closing of the channel is not complete (see also E). E, top, closing of p.T80D channel at low voltage. The bottom traces represent time scale-expanded recordings of the top trace showing insertion (left trace) and closing (right trace) events. The closing event is marked by an asterisk. Bottom, amplitude histogram derived from the recording shown at the top. The low conductance state registered after closing of the channel at +20 mV is marked by arrowheads. Arrow, counts registered at +20 mV for the channel at the fully open state. F, current-voltage dependence of the p.T80D channel at low conductance state that persisted after partial closing of the channel's pore (see D and E). G, current-voltage relationships of a mutant channel at the fully open (1) and the low conductance (2) states; n = 5–7. Error bars, S.D.

FIGURE 9.

Electrophysiological analysis of MPV17 protein with mutation p.T80D or p.T80A at different redox conditions. A, MCR showing insertion of the p.T80D channels at fully open and low conductance (marked by asterisks) states. Electrolyte conditions (A and B) were asymmetric: 1.0 m KCl trans/0.5 m KCl cis, pH 7.2, with no addition of DTT. B, current-voltage dependence of the p.T80D channel at fully open (1) and low conductance (2) states. Note that ion selectivity of the channel is the same at both states. C, current trace of the p.T80D channel pretreated with 0.2 mm H₂O₂ in response to the indicated voltage ramp protocol. Electrolyte (C and D) was symmetrical 1.0 m KCl, pH 7.2, containing no DTT. Note the complete closing (marked by an asterisk) of the channel. D, voltage-dependent open probability (P_open) of p.T80D channel pretreated (1) or not treated (2) with H₂O₂ (0.2 mm) (n = 3–6). E, current traces of the single p.T80D channel at the indicated voltages. Electrolyte conditions were symmetric (1.0 m KCl, pH 7.2) with the exception of conditions at zero holding potential (1.0 m KCl trans/0.5 m KCl cis, pH 7.2). All samples contained 10 mm DTT. The bottom trace under recording at +10 mV shows an insertion event at higher time scale resolution. The subconductance state visible during recording at −10 mV is marked by an asterisk. F, recording of the single p.T80A channel at ±40 mV. Electrolyte was symmetrical 1.0 m KCl containing 10 mm DTT and buffered with 10 mm Tris-Cl, pH 7.2. The bottom trace represents time scale-expanded recording of the top trace. Note the partial closing of the channel with the appearance of four subconductance states (numbered at the bottom trace). G, voltage-dependent open probability (P_open) of p.T80A channel (n = 4–6). Electrolyte was symmetrical 1.0 m KCl, pH 7.2, without DTT (1) or containing 10 mm DTT (2). Error bars, S.D.

FIGURE 10.

SCA of MPV17 protein with mutations p.P98L or p.C99A. A, current trace of the p.P98L channel in response to the indicated voltage ramp protocol. Electrolyte (A and B) was symmetrical 1.0 m KCl, pH 7.2, containing 10 mm DTT. Note the only partial closing (marked by asterisks in A and B) of the channel. B, current-voltage dependence of the p.P98L channel. C, voltage-dependent open probability (P_open) of the p.P98L channel registered in the presence (1) or absence (2) of 10 mm DTT. D, current-voltage dependence of two p.C99A channels according to the indicated voltage ramp protocol. The bottom trace represents a time scale-expanded recording of the top trace showing the appearance of subconductance state (marked by asterisks) during closing of one of the channels. Electrolyte (D and E) was symmetrical 1.0 m KCl, pH 7.2, containing no DTT. E, current trace of the single p.C99A channel in response to the indicated voltage ramp protocol. Protein sample was pretreated with 0.2 mm H₂O₂. F, voltage-dependent open probability (P_open) of the p.C99A channel. Electrolyte was symmetrical 1.0 m KCl, pH 7.2, without DTT. Protein samples were not treated (1) or were pretreated (2) with 0.2 mm H₂O₂. Error bars, S.D.

FIGURE 11.

Analysis of mitochondrial membrane potential and ROS formation in embryonic fibroblasts. A, average TMRE fluorescence values of fibroblasts from wild-type and Mpv17^−/− mice. Fluorescence intensity (A, C, E, and F) was detected by plate reading. *, p < 0.001; n = 6. 30 nm TMRE and 20 μm FCCP were used. B, live cell fluorescence microscopy of embryonic fibroblasts incubated with 30 nm TMRE. Note the low TMRE fluorescence of cells treated with 20 μm FCCP. Scale bar, 20 μm. C, dependence of the fluorescence values on TMRE concentration. Light and dark bars, results from wild-type and Mpv17^−/− fibroblasts, respectively. *, p < 0.05; **, p < 0.01; n = 4. D, estimation of Δψ_m using digital image analysis (see “Experimental Procedures” for details). See the legend to Fig. 11A for the conditions used. *, p < 0.05; n = 4. E, average DCF-DA and TMRE fluorescence values in fibroblasts from wild-type and Mpv17^−/− mice; effect of antioxidants: vitamin C (Vit.C; 100 μm) and N-acetyl-l-cysteine (NAC; 10 mm). Fibroblasts from the same stocks were used for all measurements. Note the increase in the level of DCF-DA fluorescence in Mpv17^−/− fibroblasts, indicating excessive ROS production. Formation of ROS was severely suppressed by antioxidants (left). However, the antioxidants did not strongly affect the levels of Δψ_m (right). *, p < 0.001 (wild-type versus Mpv17^−/− fibroblasts); #, p < 0.001 (control versus antioxidant-treated samples); n = 4. 30 nm TMRE and 10 μm DCF-DA were used. F, effect of H₂O₂ on Δψ_m of embryonic fibroblasts. The fluorescence was detected 4 h after a single injection of 50 μm H₂O₂ (top) or 48 h after injection of 200 μm H₂O₂ (middle). For chronic treatment of fibroblasts, 200 μm H₂O₂ was injected three times, every 5th day in a row and fluorescence measurements were conducted 48 h after the last injection (bottom). Cells immortalized after passage 5 were used. *, p < 0.05; **, p < 0.01; n = 6. Error bars, S.D.

FIGURE 12.

Effect of H₂O₂ treatment on morphology of mitochondria. A, representative images of fibroblasts with tubular (1) or completely (2) or partially (3) fragmented mitochondria. A higher magnification image of the boxed area in 3 is also shown (4). The cells were stained with 200 nm TMRE; scale bar, 20 μm. B and C, quantitative estimation of the effects of single (B) and chronic (C) H₂O₂ treatment (200 μm) on morphology of mitochondria (see legend to Fig. 11F for conditions of H₂O₂ treatment). The percentage of cells belonging to the corresponding morphological classification (see Fig. 12A) is shown: tubular (dark gray), partially fragmented (light gray), or completely fragmented (gray) mitochondria. 200 cells were scored for each group. Embryonic fibroblasts were immortalized after passage 5. D, semiquantitative analysis of mitochondrial morphology. The shape of mitochondria was detected according to Ref. (see “Experimental Procedures” for details). Fibroblasts from wild-type and Mpv17^−/− mice were used. Some samples (marked as +H₂O₂) were analyzed 4 h after single injection of H₂O₂ (50 μm). 120–140 cells were characterized for each group. p values are indicated; n = 3. Error bars, S.D.

FIGURE 13.

Immunodetection of mitochondrial and cytosolic proteins. A, PINK1 and LC3 proteins were detected in lysates of fibroblasts before (control) and after single or chronic H₂O₂ (200 μm) treatment (see the legend to Fig. 11F for details). Mitochondrial inner (SDH) and outer (VDAC) membrane proteins and cytosolic α-tubulin were analyzed as positive controls. B, immunodetection of the inner mitochondrial membrane protein ATP synthase subunit b (ATP synthase) in lysates of fibroblasts before (Control) and after (+H₂O₂) single H₂O₂ (200 μm) treatment of cells (see legend to Fig. 11F for details). C, hypothetical composition of the MPV17 channel interior. Only one α-helix (amino acids 80–99) of three identical amphipathic helices donated by each subunit of the homotrimeric MPV17 protein is shown. In addition to these helices, other parts of the protein molecule may participate in formation of the channel's pore. Amino acids forming the hydrophobic part of the helix (see Fig. 1, B and C) are not shown. The aspartic acid, Asp-92, responsible for ion selectivity of the channel is marked by an arrowhead. Locations of mutations leading to MDDS are marked by asterisks.