Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i. e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulat ion. Until recently, others and we have used immuno-topochemical and bioche mical methods to demonstrate the integration of the channel into the cell m embrane and endoplasmic reticulum of vertebrate cells. In the present study , we used molecular biological methods to induce the heterologous expressio n of tagged human type-1 porin in oocytes of Xenopus laevis and to illustra te its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminu s of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indica ting a strong influence of the porin N-terminus on protein trafficking to t he plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. H ere, plasmalemmal expression was observed using anti-FLAG(TM) M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver en hancement through scanning electron microscopy. In contrast to the EGFP-por in fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plas ma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are ess ential for future electrophysiological studies on the channel. (C) 2000 Els evier Science Ltd. All rights reserved.