Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel‐Forming Peptides Zervamicin, Alamethicin, and Gramicidin

Abstract

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.

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

“…Further increasing the amount of 1 applied to cells (10 μM) led to the rapid onset of linear leak currents, which agreed with results for the calcein leakage assay. Similar results were also observed in the MIA PaCa-2 cell line and are consistent with the known voltage dependence and pore-forming properties of the peptaibol alamethicin, which has long been used as a model compound for studying conductance through voltage-gated ion channels (56). Therefore, while 1 was clearly able to enter cells at cytotoxic concentrations without causing visible disruption to membranes in AFM and calcein release studies, the acute ion leakage initiated in patch-clamp studies, at its minimum cytotoxic concentration, suggested that 1 still retained a certain degree of plasma membrane-disrupting capacity, which likely contributed in part to its cytotoxic capabilities.…”

Fusaricidin-Type Compounds Create Pores in Mitochondrial and Plasma Membranes of Mammalian Cells

“…Melittin, a cationic 2.6 kDa peptide from the Apis mellifera bee venom, is hemolytic and cytotoxic starting from ~0.5 µM (1.3 µg/mL), with maximum activity at ~7 and 20 µM (18 and 52 µg/mL), respectively [31,32]. The peptaibols alamethicin and zervamicin permeabilize plasma membrane of mammalian somatic cells at concentrations of ~5 µM (10 µg/mL) [33]. Other peptides display the membrane activity and toxicity at comparable or higher concentrations [34].…”

An electrical microenvironment constructed based on electromagnetic induction stimulates neural differentiation

“…Among them, undifferentiated PC12 cells were round cells with a halo and differentiated cells became the shape of sympathetic neurons and grew neurites. 55 By analyzing neurite outgrowth, it could be found that cells on all groups had neurite, and the cells cultured in NH 2 -MXene/PLLA under a rotating magnetic field possessed the longest neurite, indicating its excellent differentiation performance. 56 The percentage and neurite length of differentiated PC12 were determined from optical images.…”