Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells
Characterisation of Nav types endogenously expressed in human SH-SY5Y neuroblastoma cells
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DOI：
10.1016/j.bcp.2012.02.022
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发表时间：
2012-06-01
影响因子：
5.8
通讯作者：
Lewis, Richard J.
中科院分区：
文献类型：
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作者：
Vetter, Irina;Mozar, Christine A.;Lewis, Richard J.
人神经母细胞瘤细胞系SH-SY5Y是鉴定和表征Na-v调节剂的潜在有用模型，但对其内源性表达Na(v)s的药理学知之甚少。本研究的目的是利用PCR和免疫组织化学方法确定SH-SY5Y细胞中内源性Na-v α和β亚基的表达，并利用电生理和荧光方法对该细胞系内源性Na-v亚型进行药理学表征。SH-SY5Y人神经母细胞瘤细胞内源性表达Na(v)1.2和Na(v)1.7等几种Na-v亚型。用缬草碱或蝎子毒素001激活内源性表达的Na(v)s引起膜去极化和随后的Ca2+通过电压门控的L型和n型钙通道内流，允许用膜电位和荧光Ca-2染料检测Na-v激活。mu-Conotoxin TIIIA和ProTxII鉴定Na(v)1.2和Na(v)1.7是这种反应的主要贡献者。Na(v)1.7选择性蝎子毒素OD1与veratridine联合产生Na(v)1.7选择性反应，证实了SH-SY5Y细胞内源性表达的人Na(v)1.7具有功能性和协同激活作用，为配体筛选提供了一种新的检测格式。爱思唯尔公司版权所有版权所有。
The human neuroblastoma cell line SH-SY5Y is a potentially useful model for the identification and characterisation of Na-v modulators, but little is known about the pharmacology of their endogenously expressed Na(v)s. The aim of this study was to determine the expression of endogenous Na-v alpha and beta subunits in SH-SY5Y cells using PCR and immunohistochemical approaches, and pharmacologically characterise the Na-v isoforms endogenously expressed in this cell line using electrophysiological and fluorescence approaches. SH-SY5Y human neuroblastoma cells were found to endogenously express several Na-v isoforms including Na(v)1.2 and Na(v)1.7. Activation of endogenously expressed Na(v)s with veratridine or the scorpion toxin 001 caused membrane depolarisation and subsequent Ca2+ influx through voltage-gated L- and N-type calcium channels, allowing Na-v activation to be detected with membrane potential and fluorescent Ca-2 dyes. mu-Conotoxin TIIIA and ProTxII identified Na(v)1.2 and Na(v)1.7 as the major contributors of this response. The Na(v)1.7-selective scorpion toxin OD1 in combination with veratridine produced a Na(v)1.7-selective response, confirming that endogenously expressed human Na(v)1.7 in SH-SY5Y cells is functional and can be synergistically activated, providing a new assay format for ligand screening. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.