Clustering of plasma membrane-bound cytochrome b5 reductase within 'lipid raft' microdomains of the neuronal plasma membrane.

Plasma membrane redox centres play a major role in neuronal defence against oxidative stress and survival. In cerebellar granule neurons in culture (CGN) a large pool of the flavoproteins are associated with the plasma membrane, and the intensity of CGN green/orange autofluorescence correlated with the levels of expression of cytochrome b(5) reductase. Regionalization of cytochrome b(5) reductase in the plasma membrane of CGN by fluorescence resonance energy transfer points out the close proximity between cytochrome b(5) reductase and the 'lipid raft' markers cholera toxin B and caveolin-2. This study unravels that membrane-bound cytochrome b(5) reductase is largely enriched at interneuronal contact sites in the neuronal soma and associated with 'lipid rafts' of the CGN plasma membrane. We also show that cytochrome b(5) reductase makes a large contribution to the NADH oxidase activity and to the red-shifted flavine fluorescence of purified rat brain synaptic plasma membranes. In conclusion, membrane-bound cytochrome b(5) reductase forms a large mesh of redox centres associated with the neuronal plasma membrane.

Modulation of sarcoplasmic reticulum Ca(2+)-ATPase by chronic and acute exposure to peroxynitrite.

The Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SERCA), an integral membrane protein, becomes irreversibly inactivated in vitro by the addition of a single bolus of peroxynitrite with a K(0.5) of 200-300 microm, and this results in a large decrease of the ATP-dependent Ca2+ gradient across the sarcoplasmic reticulum (SR) membranes. The inactivation of SERCA is raised by treatment of SR vesicles with repetitive micromolar pulses of peroxynitrite. The inhibition of the SERCA is due to the oxidation of thiol groups and tyrosine nitration. Scavengers that react directly with peroxynitrite, such as cysteine, reduced glutathione, NADH, methionine, ascorbate or Trolox, a water-soluble analog of alpha-tocopherol, afforded significant protection. However, dimethyl sulfoxide and mannitol, two hydroxyl radical scavengers, and alpha-tocopherol did not protect SERCA from inactivation. Our results showed that the target of peroxynitrite is the cytosolic globular domain of the SERCA and that major skeletal muscle intracellular reductants (ascorbate, NADH and reduced glutathione) protected against inhibition of this ATPase by peroxynitrite.