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.

Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects

“…In normal situations, ⌬⌿ m is maintained mainly through a balance between transmembrane proton pumping to the intermembrane space mediated by electron transfer and proton translocation to the matrix for ATP synthesis by the ATPase complex. A number of studies have examined ⌬⌿ m in mitochondrial disease, with various reports revealing lower values compared with controls and other reports describing conditions in which a higher ⌬⌿ m was found (2,13,41,47). Alterations in ⌬⌿ m are variable and dependant on the type of mitochondrial mutation.…”

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

“…Among the possible consequences of ATP6 point mutations, impaired F 1 F 0 assembly (26,27), enzyme uncoupling (11,40), and proton conduction blockade through F 0 (6, 7, 9) have all been proposed. To distinguish between them, concluding evidence on the effect of NARP/MILS mutations on F 1 F 0 assembly was limited because the reported human F 1 F 0 structural studies have been done mostly with heteroplasmic cells and provided apparently opposing results (9,(25)(26)(27)).…”

“…Oligomycin Sensitivity in T8993G and T8993C MutantsBased on studies on heteroplasmic T8993G cells, some groups (11,40) propose that the pathogenic mechanism of NARP/ MILS is a partial uncoupling of F 0 -proton translocation from F 1 -ATP synthesis. We evaluated functional coupling between F 1 and F 0 sectors in the homoplasmic T8993G cybrids by assaying oligomycin sensitivity during ATP synthesis and hydrolysis (Fig.…”

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

“…The mutant protein imparts upon F 1 F 0 -ATPase a defect in ATP synthesis, but not hydrolysis, 11 which may result from inefficient coupling between proton transport and ATP synthesis. 12 At the DNA level, the T8993G transversion generates a unique recognition site for SmaI and XmaI restriction endonucleases (REs), which is absent in wild type mtDNA and can be used for the diagnostic purposes. In vitro, REs digest up to 99.95% of the corresponding recognition sites, as estimated by PCR, 13 making the mitochondrial targeting of the SmaI or XmaI REs an attractive therapeutic strategy for the treatment of NARP and MILS syndromes.…”