Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23
Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23
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DOI：
10.1016/j.neuropharm.2003.09.018
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发表时间：
2004-03-01
影响因子：
4.7
通讯作者：
Trezise, DJ
中科院分区：
文献类型：
--
作者：
John, VH;Main, MJ;Trezise, DJ
电压门控钠通道Na（v）1.8（SNS，PN 3）被认为是背根神经节（DRG）河豚毒素抗性（TTX-R）Na+电流的分子相关物。TTX-R/Na（v）1.8是一个有吸引力的治疗药物靶点，用于炎症和神经病理性疼痛的基础上，其特异性分布在感觉神经元和炎症介质的调节。然而，在哺乳动物细胞中稳定表达功能蛋白的困难阻碍了对重组Na（v）1.8的详细分析。在这里，我们显示了大鼠Na（v）1.8（rNa（v）1.8）在大鼠DRG/小鼠N18 Tg 2神经母细胞瘤杂交瘤细胞系ND 7 -23中的稳定表达和功能分析。记录了大鼠Na（v）1.8 Na+电流（789 +/- 89 pA，n = 62，超过20代细胞），其在门控动力学和激活和失活的电压依赖性方面定性类似于DRG TTX-R。局部麻醉药丁卡因产生rNa（v）1.8的紧张性抑制（平均IC 50值12.5 μ M），并且在重复门控范例（2-10 Hz）中也显示频率依赖性阻滞。抗惊厥镇痛药拉莫三嗪的几种类似物抑制TTX-R的能力与rNa（v）1.8相关（r = 0.72，P < 0.001）。RT-PCR分析野生型ND 7 -23细胞显示内源性表达β 1和β 3辅助Na+通道亚单位，并讨论了这些亚单位的存在辅助和稳定rNa（v）1.8表达的可能性。我们的结论是，神经母细胞瘤ND 7 -23细胞系是rNa（v）1.8 Na+的合适异源表达系统通道，因为它允许具有生物物理特性的通道的稳定表达，所述生物物理特性非常类似于DRG神经元中的天然TTX-R电流。该试剂可用于寻找rNa（v）1.8的药理学抑制剂作为新型镇痛剂。(C)2003 Elsevier Ltd.保留所有权利。
The voltage-gated sodium channel Na(v)1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/Na(v)1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant Na(v)1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells.Here, we show stable expression and functional analysis of rat Na(v)1.8 (rNa(v)1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat Na(v)1.8 Na+ currents were recorded (789 +/- 89 pA, n = 62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNa(v)1.8 (mean IC50 value 12.5 muM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNa(v)1.8 (r = 0.72, P < 0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta 1 and beta 3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNa(v)1.8 is discussed.We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNa(v)1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove Useful in the search for pharmacological inhibitors of rNa(v)1.8 as novel analgesics. (C) 2003 Elsevier Ltd. All rights reserved.