RECONSTITUTION OF THE NATIVE MITOCHONDRIAL OUTER-MEMBRANE IN PLANAR BILAYERS - COMPARISON WITH THE OUTER-MEMBRANE IN A PATCH PIPETTE AND EFFECT OF ALUMINUM COMPOUNDS

Detergent-free rat brain outer mitochondrial membranes were incorporat ed in planar lipid bilayers in the presence of an osmotic gradient, an d studied at high (1 m KCl) and low (150 mm KCl) ionic strength soluti ons. By comparison, the main outer mitochondrial membrane protein, VDA C, extracted from rat liver with Triton X-100, was also studied in 150 mm KCl. In 1 m KCl, brain outer membranes gave rise to electrical pat terns which resembled very closely those widely described for detergen t-extracted VDAC, with transitions to several subconducting states upo n increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, how ever, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mm KCl, bilayers containing only one channel had a conductance of 700 +/- 23 pS for rat brain outer me mbranes, and 890 +/- 29 pS for rat liver VDAC. Use of a fast time reso lution setup allowed demonstration of open-close transitions in the mi llisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference highe r than approx. +/- 60 mV induced an almost irreversible decrease of th e single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the be havior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Bioph ys. J. 20:311-319). The neurotoxicological action of aluminum was stud ied in single outer membrane channels from rat brain mitochondria. We found that Am concentrations of Al Cl3 and aluminum lactate decreased the conductance by about 50%, when the applied potential difference wa s positive relative to the side of the metal addition.