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

Gianluca Sgarbi, Alessandra Baracca, Giorgio Lenaz, Lucia M. Valentino, Valerio Carelli, Giancarlo Solaini

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Pages (from-to)493-500
Number of pages8
JournalBiochemical Journal
Volume395
Issue number3
DOIs
Publication statusPublished - May 1 2006

Fingerprint

Leigh Disease
Mitochondrial DNA
Protons
Rhodamine 123
Adenosine Triphosphate
Mutation
Adenosine Triphosphatases
Quenching
Genes
Fluxes
Oligomycins
Mitochondria
Proton conductivity
Lymphocytes
Enzymes
Fluorescent Dyes
Catalysis
Nervous System Diseases
Point Mutation
Fluorescence

Keywords

  • ATPase 6 subunit
  • FF ATPase
  • Membrane potential
  • Mitochondria
  • Mitochondrial DNA (mtDNA) point mutation
  • Proton transport

ASJC Scopus subject areas

  • Biochemistry

Cite this

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. / Sgarbi, Gianluca; Baracca, Alessandra; Lenaz, Giorgio; Valentino, Lucia M.; Carelli, Valerio; Solaini, Giancarlo.

In: Biochemical Journal, Vol. 395, No. 3, 01.05.2006, p. 493-500.

Research output: Contribution to journalArticle

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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.",
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