RECONSTITUTION OF THE NATIVE MITOCHONDRIAL OUTER-MEMBRANE IN PLANAR BILAYERS - COMPARISON WITH THE OUTER-MEMBRANE IN A PATCH PIPETTE AND EFFECT OF ALUMINUM COMPOUNDS
T. Mirzabekov et al., RECONSTITUTION OF THE NATIVE MITOCHONDRIAL OUTER-MEMBRANE IN PLANAR BILAYERS - COMPARISON WITH THE OUTER-MEMBRANE IN A PATCH PIPETTE AND EFFECT OF ALUMINUM COMPOUNDS, The Journal of membrane biology, 133(2), 1993, pp. 129-143
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.