Membrane permeabilization of a mammalian neuroendocrine cell type (PC12) by the channel-forming peptides zervamicin, alamethicin, and gramicidin.

Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram-positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1 muM GRAM addition to PC12 cells kept at a membrane potential V(m)=0 mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. -24 mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5-10 microM concentrations while V(m) is kept at -60 mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1-2 min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10-20 min at V(m)=0 mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K(+)-release measurements from a PC12 suspension with a K(+)-selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis-positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell.