Isolation of mitochondrial porin of the fly Protophormia: porin modification by the pesticide CGA 140'408 studied in lipid bilayer membranes.
Mitochondrial porin from the fly Protophormia was solubilized with detergent from whole mitochondria and purified by chromatography across a hydroxyapatite (HPT) column. The purified protein had an apparent molecular mass of about 30 kDa on SDS-PAGE. Partial sequencing of the protein confirmed that it is porin. When reconstituted in planar lipid bilayer membranes, porin formed ion-permeable channels with single-channel conductances of 2.4 and 4.5 nS in 1 M KCl. At low voltage, Protophormia porin displayed the properties of a general diffusion pore and had a small selectivity for anions over cations. At transmembrane potentials starting with about 20-30 mV, the channel switched in closed state, which is still ion-permeable. Our results suggest that Protophormia porin possesses functional properties similar to those of other mitochondrial porins. Porin was also isolated and purified from mitochondria, which were treated with the carbodiimide CGA 140'408 It represents the active derivative of diafenthiuron a new acaricide and insecticide. This carbodiimide labels both a F0-component of the inner membrane ATPase and outer membrane porin in a similar way as N,N'-dicyclohexylcarbodiimide (DCCD). Reconstitution experiments with the CGA 140'408-modified porin showed no significant effect of the modification on the single-channel conductance, suggesting that CGA 140'408 binds outside the channel. The voltage-dependence of the CGA 140'408-modified porin was changed with respect to the unmodified form. The closed configuration of the pesticide-modified channel was reached at smaller transmembrane potentials, suggesting a shift of the open to the closed state of Protophormia porin by pesticide binding. A possible contribution of this effect to the pesticide action is discussed.
Wiesner
P
P
Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum), Universität Würzburg, Germany.
Popp
B
B
Schmid
A
A
Benz
R
R
Kayser
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D004175:Diptera / Q000648:ultrastructure; D004553:Electric Conductivity; D007306:Insecticides / Q000494:pharmacology*; D007473:Ion Channels / Q000502:physiology; D008051:Lipid Bilayers / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D018272:Porins; D017421:Sequence Analysis; D050994:Voltage-Dependent Anion Channels
D002234:Carbodiimides; D007306:Insecticides; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018272:Porins; D050994:Voltage-Dependent Anion Channels; C087660:CGA 140408