Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode.
Electroporation followed by electrochemical measurement of quantal transmitter release from single cells using a patterned microelectrode.
复制标题
DOI：
10.1039/c3lc41324a
复制
发表时间：
2013-06-07
期刊：
影响因子：
6.1
通讯作者：
Gillis KD
中科院分区：
文献类型：
--
作者：
Ghosh J;Liu X;Gillis KD
紧邻单个神经内分泌细胞的电化学微电极可以记录由单个囊泡的可氧化递质胞吐（即量子胞吐）引起的安培电流峰值。在这里，我们报告了一种有效方法的发展，该方法使用相同的电化学微电极对相邻的染色质细胞进行电渗透，然后使用安培法测量随之而来的儿茶酚胺的量子释放。电压脉冲序列，振幅为5 - 7v，持续时间为0.1-0.2 ms，用于可靠地触发使用金电极的细胞释放。电渗透引起的电流峰值与去极化高K+溶液引起的电流峰值具有相似的面积、峰高和持续时间，因此释放发生在单个分泌颗粒中。细胞对台盼蓝染色的摄取表明，电压刺激使质膜通透。电压脉冲不会降低使用测试分析物进行分析的电极的电化学灵敏度。令人惊讶的是，在没有Ca2+的溶液（0 Ca2+/5 mM EGTA）中，电穿孔引起了强大的量子释放。相比之下，电渗透诱导的递质释放需要在槽液中使用Cl -，在2到32 mM之间的实验中，电渗透诱导的递质释放速率对[Cl -]有很大的依赖性。使用相同的电化学电极电穿孔并记录单个染色质细胞中儿茶酚胺的量子释放，可以精确地定时刺激，一次刺激单个细胞，并可用于将膜外物质装载到细胞中。
An electrochemical microelectrode located immediately adjacent to a single neuroendocrine cell can record spikes of amperometric current that result from exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. Here, we report the development of an efficient method where the same electrochemical microelectrode is used to electropermeabilize an adjacent chromaffin cell and then measure the consequent quantal catecholamine release using amperometry. Trains of voltage pulses, 5–7 V in amplitude and 0.1–0.2 ms in duration, were used to reliably trigger release from cells using gold electrodes. Amperometric spikes induced by electropermeabilization had similar areas, peak heights and durations as amperometric spikes elicited by depolarizing high K+ solutions, therefore release occurs from individual secretory granules. Uptake of trypan blue stain into cells demonstrated that the plasma membrane is permeabilized by the voltage stimulus. Voltage pulses did not degrade the electrochemical sensitivity of the electrodes assayed using a test analyte. Surprisingly, robust quantal release was elicited upon electroporation in the absence of Ca2+ in the bath solution (0 Ca2+/5 mM EGTA). In contrast, electropermeabilization-induced transmitter release required Cl− in the bath solution in that bracketed experiments demonstrated a steep dependence of the rate of electropermeabilization-induced transmitter release on [Cl−] between 2 and 32 mM. Using the same electrochemical electrode to electroporate and record quantal release of catecholamines from an individual chromaffin cell allows precise timing of the stimulus, stimulation of a single cell at a time, and can be used to load membrane-impermeant substances into a cell.
登录
查看更多内容
影响因子：
6.1
作者：
Dittami GM;Rabbitt RD
通讯作者：
Rabbitt RD
影响因子：
7.4
作者：
CAHILL, PS;WIGHTMAN, RM
通讯作者：
WIGHTMAN, RM
影响因子：
3
作者：
Segura, F;Brioso, MA;Borges, R
通讯作者：
Borges, R
影响因子：
2.4
作者：
KNIGHT, DE;BAKER, PF
通讯作者：
BAKER, PF
影响因子：
3
作者：
Spegel, Christer;Heiskanen, Arto;Ruzgas, Tautgirdas
通讯作者：
Ruzgas, Tautgirdas