Schledermann W, Wulfsen I, Schwarz J R, Bauer C K
Abteilung für Angewandte Physiologie, Institut für Physiologie, Universitätsklinikum Hamburg-Eppendorf, Universität Hamburg, D-20246 Hamburg, Germany.
J Physiol. 2001 Apr 1;532(Pt 1):143-63. doi: 10.1111/j.1469-7793.2001.0143g.x.
The mechanism of thyrotropin-releasing hormone (TRH)-induced ether-a-go-go-related gene (erg) K+ current modulation was investigated with the perforated-patch whole-cell technique in clonal somatomammotroph GH3/B6 cells. These cells express a small endogenous erg current known to be reduced by TRH. GH3/B6 cells were injected with cDNA coding for rat erg1, erg2, erg3 and HERG K+ channels. The corresponding erg currents were isolated with the help of the specific erg channel blockers E-4031 and dofetilide and their biophysical properties were determined. TRH (1 M) was able to significantly reduce the different erg currents. The voltage dependence of activation was shifted by 15 mV (erg1), 10 mV (erg2) and 6 mV (erg3) to more positive potentials without strongly affecting erg inactivation. TRH reduced the maximal available erg current amplitude by 12% (erg1), 13% (erg2) and 39% (erg3) and accelerated the time course of erg1 and erg2 channel deactivation, whereas erg3 deactivation kinetics were not significantly altered. The effects of TRH on HERG currents did not differ from those on its rat homologue erg1. In addition, coinjection of rat MiRP1 with HERG cDNA did not influence the TRH-induced modulation of HERG channels. Rat erg1 currents recorded in the cell-attached configuration were reduced by application of TRH to the extra-patch membrane in the majority of the experiments, confirming the involvement of a diffusible second messenger. Application of the phorbol ester phorbol 12-myristate 13-acetate (PMA; 1 M) shifted the voltage dependence of erg1 activation in the depolarizing direction, but it did not reduce the maximal current amplitude. The voltage shift could not be explained by a selective effect on protein kinase C (PKC) since the PKC inhibitor bisindolylmaleimide I did not block the effects of TRH and PMA on erg1. In addition, cholecystokinin, known to activate the phosphoinositol pathway similarly to TRH, did not significantly affect the erg1 current. Various agents interfering with different known TRH-elicited cellular responses were not able to completely mimic or inhibit the TRH effects on erg1. Tested substances included modulators of the cAMP-protein kinase A pathway, arachidonic acid, inhibitors of tyrosine kinase and mitogen-activated protein kinase, sodium nitroprusside and cytochalasin D. The results demonstrate that all three members of the erg channel subfamily are modulated by TRH in GH3/B6 cells. In agreement with previous studies on the TRH-induced modulation of the endogenous erg current in prolactin-secreting anterior pituitary cells, the TRH effects on overexpressed erg1 channels are not mediated by any of the tested signalling pathways.
采用穿孔膜片全细胞技术，在克隆的生长激素释放激素分泌细胞GH3/B6中研究促甲状腺激素释放激素（TRH）诱导的醚 - 去极化相关基因（erg）钾电流调节机制。这些细胞表达少量内源性erg电流，已知该电流会被TRH降低。向GH3/B6细胞注射编码大鼠erg1、erg2、erg3和HERG钾通道的cDNA。借助特异性erg通道阻滞剂E - 4031和多非利特分离出相应的erg电流，并测定其生物物理特性。TRH（1 μM）能够显著降低不同的erg电流。激活的电压依赖性分别向更正电位偏移15 mV（erg1）、10 mV（erg2）和6 mV（erg3），而对erg失活影响不大。TRH使最大可用erg电流幅度降低12%（erg1）、13%（erg2）和39%（erg3），并加速erg1和erg2通道失活的时间进程，而erg3失活动力学未发生显著改变。TRH对HERG电流的影响与其对大鼠同源物erg1的影响无差异。此外，将大鼠MiRP1与HERG cDNA共注射并不影响TRH诱导的HERG通道调节。在大多数实验中，向贴附细胞模式下记录的大鼠erg1电流的膜外施加TRH可使其降低，证实了可扩散第二信使的参与。佛波酯佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA；1 μM）使erg1激活的电压依赖性向去极化方向偏移，但未降低最大电流幅度。该电压偏移不能用对蛋白激酶C（PKC）的选择性作用来解释，因为PKC抑制剂双吲哚基马来酰亚胺I并未阻断TRH和PMA对erg1的作用。此外，已知与TRH类似激活磷脂酰肌醇途径的胆囊收缩素对erg1电流无显著影响。各种干扰不同已知TRH引发的细胞反应的试剂均不能完全模拟或抑制TRH对erg1的作用。测试物质包括cAMP - 蛋白激酶A途径调节剂、花生四烯酸、酪氨酸激酶和丝裂原活化蛋白激酶抑制剂、硝普钠和细胞松弛素D。结果表明，erg通道亚家族的所有三个成员在GH3/B6细胞中均受TRH调节。与先前关于TRH诱导的催乳素分泌性垂体前叶细胞内源性erg电流调节的研究一致，TRH对过表达的erg1通道的作用并非由任何测试的信号通路介导。
