Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel‐Forming Peptides Zervamicin, Alamethicin, and Gramicidin
Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel‐Forming Peptides Zervamicin, Alamethicin, and Gramicidin
复制标题
通道形成肽 Zervamicin、Alamethicin 和 Gramicidin 对哺乳动物神经内分泌细胞类型 (PC12) 的膜通透作用
作者：
A. Weidema;T. Kropacheva;J. Raap;D. Ypey
Zervamicin IIB(ZER)是一种16聚肽，在人造膜中产生电压依赖的电导，这一特性被认为是其主要对革兰氏阳性微生物具有抗菌活性的原因。此外，ZER似乎抑制了小鼠的运动活动(见本期其他部分)，可能是通过影响大脑。为了检测中枢神经系统神经细胞的电生理特性是否可能受到成孔ZER的影响，本研究首次试图揭示这种生物学活性的分子机制。为此，用全细胞膜片钳技术研究了通道形成的ZER对神经元样大鼠嗜铬细胞瘤细胞(PC12)的膜通透性，并与著名的电压门控性多肽Alamethicin F50/5(ALA)和阳离子通道形成肽-抗生素Granicidin D(Gram)的通透性进行了比较。在保持膜电位Vm=0 μmV的PC12细胞中加入1 M GRAM可导致漏电导无延迟地逐渐增加，其负反转电位约为−24 mV，ZER和ALA在该浓度和电位下无效。然而，如果添加5-10 μM浓度的ZER和ALA，而Vm保持在−60 mV，它们会在延迟1-2 分钟后引起PC12细胞膜突然而强烈的渗透，在该时间尺度上通常会导致贴片细胞的崩解形态变化，但不会导致周围细胞的崩解形态变化。建立的电导的零反转电势与所形成的通道的已知的非选择性一致。这种突然的通透性在Vm=0 mV时不会在10-20 分钟内发生，这与人造脂膜中ZER和ALA通道形成的已知电压依赖性一致。PC12悬浮液中的K+释放测量进一步支持了这些肽醇对整个培养物的渗透作用。进一步的分析表明，这种通透作用与细胞外或细胞内钙效应有关，因为Ba2+抑制了ZER和ALA的通透作用。我们的结论是，对于哺乳动物神经元样PC12细胞膜，ZER和ALA多肽的通透作用不同于GRAN，这与早期在其他(人工)膜系统中对这些多肽的研究一致。它们在生理范围内由顺式正膜电位增加，可能包括钙进入PC12细胞。
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 μM GRAM addition to PC12 cells kept at a membrane potential Vm=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 μM concentrations while Vm 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 Vm=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.
影响因子：
56.9
作者：
KETCHEM, RR;HU, W;CROSS, TA
通讯作者：
CROSS, TA