Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.
Effects of 4,9-anhydrotetrodotoxin on voltage-gated Na+ channels of mouse vas deferens myocytes and recombinant NaV1.6 channels.
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4,9-脱水河豚毒素对小鼠输精管肌细胞电压门控 Na 通道和重组 NaV1.6 通道的影响。
DOI：
10.1007/s00210-018-1476-6
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Teramoto N
中科院分区：
文献类型：
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作者：
Takahara K;Yamamoto T;Uchida K;Zhu HL;Shibata A;Inai T;Noguchi M;Yotsu-Yamashita M;Teramoto N
为了确定小鼠输精管电压门控Na+通道β-亚基的主要特征，进行了分子研究。实时荧光定量聚合酶链式反应显示，Scn1b基因的表达水平显著高于其他β亚基基因(Scn2b-Scn4b)。Scn1b蛋白在小鼠输精管内环和外纵肌中也有表达。在全细胞记录中，比较了4，9-anwater TTX对心肌细胞(即天然Ina)电压门控性Na+电流峰值的作用及其对重组NaV1.6通道(在HEK293细胞中表达)的抑制作用。在去极化的矩形电压脉冲作用下，HEK293细胞中表达的重组NAV1.6(即重组INA)和天然INA发生了快速而短暂的内向。天然INA的电流衰减与与β1亚基共表达的重组NaV1.6电流相似。天然INA的电流-电压(I-V)关系与与β1亚基共表达的重组NaV1.6电流相似。4，9-脱水TTX抑制天然INA(Ki= 510nM)、重组INA(Ki= 112nM)和含有β1亚基的重组INACO表达的峰值(Ki= 92nM)。天然INAVA的半最大(VHalf)激活和失活与用β1亚基表达的重组INACO相似。这些结果表明，β1亚单位蛋白可能主要在小鼠输精管的平滑肌层中表达，4，9-脱水TTX不仅抑制自然表达的β，而且还以浓度依赖的方式抑制重组INA和与其共同表达的重组INACO。
Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+channel β-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression ofScn1bwas significantly higher than that of other β-subunit genes (Scn2b–Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INaand recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INawas similar to the recombinant NaV1.6 current co-expressed with β1-subunits. The current-voltage (I-V) relationships of native INawere similar to those of recombinant NaV1.6 currents co-expressed with β1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa(Ki= 510 nM), recombinant INa(Ki= 112 nM), and recombinant INaco-expressed with β1-subunits (Ki= 92 nM). The half-maximal (Vhalf) activation and inactivation of native INavalues were similar to those observed in recombinant INaco-expressed with β1-subunits. These results suggest that β1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INabut also recombinant INaand recombinant INaco-expressed with β1-subunits in a concentration-dependent manner.