Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA

Abstract

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.

Figure 1. Pedigree of four families carrying the mtDNA T8993G mutation and mtDNA heteroplasmy of lymphocyte probands

Top, pedigrees of the four Italian families investigated. White symbols indicate unaffected individuals without the mutation; hatched symbols, carrier of the mutation with non-specific symptoms; dark-grey symbols, individuals with full-blown NARP or MILS. Bottom, electrophoretic gel showing the AvaI digestion of PCR products. The presence of three fragments correspond to the coexistence of both wild-type (551 bp) and mutant (345 and 206 bp) mtDNA in patients. Mutant mtDNA was absent in the control samples. Relative densitometric quantification of the AvaI-digested fragments allows evaluation of the percentage of mtDNA heteroplasmy. MW, size markers

Figure 2. Mitochondrial ATP synthesis in digitonin-treated lymphocytes

The reaction mixture contained: 20×10⁶ cells/ml, 60 μg/ml digitonin, 2 mM iodoacetamide, 25 μM P¹, P⁵-di(adenosine-5′) pentaphosphate pentasodium salt, 100 mM KCl, 1 mM EGTA, 3 mM EDTA, 5 mM KH₂PO₄ and 2 mM MgCl₂ in Tris/HCl (pH 7.4). ATP synthesis was supported by adding 20 mM succinate (in the presence of 4 μM rotenone) and 0.5 mM ADP. The reaction was carried out at 30 °C for 5 min, and the amount of ATP synthesized was measured with the luciferin/luciferase chemiluminescent method [15]. Data reported for patients are presented as mean for two determinations in lymphocyte preparations.

Figure 3. RH-123 fluorescence changes in digitonin-permeabilized lymphocytes upon addition of respiratory substrates and inhibitors

(A) Fluorescence measurements were carried out in a respiratory buffer (250 mM sucrose, 10 mM Hepes, 100 μM K-EGTA, 2 mM MgCl₂ and 4 mM KH₂PO₄ (pH 7.4), containing an ADP regenerating system (10 mM glucose and 2.5 units of hexokinase). Additions were 50 nM RH-123, 2×10⁶/ml lymphocytes, 33 nM cyclosporin, 10 mM glutamate/10 mM malate, 15 μg/ml digitonin, 200 μM ADP, 2.5 μM rotenone, 20 mM succinate, 2 μM oligomycin and 1 μg/ml(1.8 μM) antimycin. (B) RH-123 fluorescence kinetics induced by the addition of 20 mM succinate to digitonin-treated lymphocytes pre-incubated or not with 2 μM oligomycin. The sample mixture consisted of 0.5 ml of respiratory buffer containing an ADP regenerating system, 50 nM RH-123, 2×10⁶/ml lymphocytes, 33 nM cyclosporin, 2.5 μM rotenone, 0.2 mM ADP and 15 μg/ml digitonin.

Figure 4. Δψ_m in lymphocytes of individuals harbouring the mtDNA T8993G mutation

Digitonin-permeabilized lymphocytes (2×10⁶/ml) incubated with 50 nM RH-123 were energized with succinate, and the kinetics of the accumulation of the positively charged probe in mitochondria was evaluated by monitoring the fluorescence emission at 527 nm (λex=503 nm). The sample mixture contained 0.5 ml of respiratory buffer [250 mM sucrose, 10 mM Hepes, 100 μM K-EGTA, 2 mM MgCl₂ and 4 mM KH₂PO₄ (pH 7.4)] with an ADP regenerating system; 33 nM cyclosporin A, 2.5 μM rotenone and 0.2 mM ADP. Patient data (state 3 respiratory condition) are reported as the mean for two measurements, whereas control data (state 3 respiratory condition) and data from both oligomycin-treated controls and patients are reported as the means±S.D.

Figure 5. Correlation between ATP synthesis rate, Δψ_m and mutation load

ATP synthesis rate and Δψ were measured in permeabilized lymphocytes from both 10 probands carrying the mtDNA T8993G mutation and 12 healthy and age-matched volunteers as controls. (A) The correlation between ATP synthesis rate and mutation load (r²=0.85). (B) The correlation between Δψ_m and mutation load (r²=0.72). (C) The correlation between ATP synthesis rate and Δψ_m (r²=0.83).

Figure 6. Ribbon diagram comparing the predicted four transmembrane α-helices of wild-type and mutant human ATPase 6

Top, the scheme is based on the three-dimensional model of the E. coli ac₁₂ complex [41] (PDB accession number, 1C17). Leu-156 of the mitochondrial enzyme is shown in yellow, whereas the essential Arg-159 and Arg-156 (in the mutant enzyme only) residues are shown in red. The amino acids reported to take part in the proton translocation [33] are represented in green. Bottom, the partial amino acid sequence of wild-type human ATPase 6. Transmembrane α-helices, as predicted by homology modelling software (EasyPred, http://www.cbs.dtu.dk/biotools/EasyPred/), are indicated by red (mutated) and black (wild-type) lines.