Department of Pharmacology, Faculty of Medicine, Saga University, Saga, 849-8501, Japan.
Department of Morphological Biology, Division of Biomedical Sciences, Fukuoka Dental College, Fukuoka, 814-0193, Japan.
Naunyn Schmiedebergs Arch Pharmacol. 2018 May;391(5):489-499. doi: 10.1007/s00210-018-1476-6. Epub 2018 Feb 16.
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 of Scn1b was 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 I) were compared with its inhibitory potency on recombinant Na1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native I and recombinant Na1.6 expressed in HEK293 cells (i.e., recombinant I). The current decay of native I was similar to the recombinant Na1.6 current co-expressed with β-subunits. The current-voltage (I-V) relationships of native I were similar to those of recombinant Na1.6 currents co-expressed with β-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native I (K = 510 nM), recombinant I (K = 112 nM), and recombinant I co-expressed with β-subunits (K = 92 nM). The half-maximal (V) activation and inactivation of native I values were similar to those observed in recombinant I co-expressed with β-subunits. These results suggest that β-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 I but also recombinant I and recombinant I co-expressed with β-subunits in a concentration-dependent manner.
为了确定小鼠输精管电压门控钠通道β亚基的主要特征，进行了分子研究。实时定量 PCR 显示 Scn1b 的表达明显高于其他β亚基基因（Scn2b-Scn4b）。Scn1b 蛋白的免疫反应性也在小鼠输精管的内环状和外纵平滑肌中被检测到。在全细胞记录中，比较了 4,9-脱水 TTX 对心肌细胞（即天然 I）电压门控 Na 电流峰值的作用与其对重组 Na1.6 通道（在 HEK293 细胞中表达）的抑制效力。去极化矩形电压脉冲引发快速和短暂的内向天然 I 和在 HEK293 细胞中表达的重组 Na1.6（即重组 I）。天然 I 的电流衰减与共表达β亚基的重组 Na1.6 电流相似。天然 I 的电流-电压（I-V）关系与共表达β亚基的重组 Na1.6 电流相似。应用 4,9-脱水 TTX 抑制了天然 I（K = 510 nM）、重组 I（K = 112 nM）和共表达β亚基的重组 I（K = 92 nM）的峰值幅度。天然 I 的半最大（V）激活和失活值与共表达β亚基的重组 I 观察到的值相似。这些结果表明，β亚基蛋白可能主要在小鼠输精管的平滑肌层中表达，并且 4,9-脱水 TTX 以浓度依赖的方式不仅抑制天然 I，而且还抑制重组 I 和共表达β亚基的重组 I。
