Ethylbromide tamoxifen, a membrane-impermeant antiestrogen, activates smooth muscle calcium-activated large-conductance potassium channels from the extracellular side
Ethylbromide tamoxifen, a membrane-impermeant antiestrogen, activates smooth muscle calcium-activated large-conductance potassium channels from the extracellular side
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DOI：
10.1124/mol.61.5.1105
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发表时间：
2002-05-01
影响因子：
3.6
通讯作者：
Sanders, KM
中科院分区：
文献类型：
--
作者：
Dick, GM;Hunter, AC;Sanders, KM
平滑肌钙激活的大电导钾通道（BK通道）被他莫昔芬和17-β-雌二醇激活。NPo的增加，通道数N乘以开放概率，取决于调节β 1亚基的存在。此外，先前的研究表明，17-β-雌二醇可能结合β 1亚基上的细胞外位点。因为他莫昔芬和17-β-雌二醇可能共享一个共同的结合位点，我们假设他莫昔芬通过膜细胞外表面的一个位点激活BK通道。合成了他莫昔芬的膜不透性类似物，乙基溴他莫昔芬，并用于在犬结肠平滑肌细胞的全细胞、由外向外、细胞附着和由内而外的贴片中测试这一假设。溴化乙锭他莫昔芬带正电荷，因此不渗透膜。在全细胞实验中，乙基溴他莫昔芬在+40 mV的正电位下增加K+电流，这以前被归因于BK通道。与他莫昔芬不同，溴乙烷他莫昔芬不抑制延迟整流电流。在由外向外的贴剂中，乙基溴他莫昔芬增加BK通道NPo，EC 50值为1 μ M。溴化乙锭他莫昔芬并没有增加BK通道NPO在细胞贴附或由内而外的补丁，但是，随后加入等摩尔他莫昔芬。这两种药物降低BK通道单一电导的程度，抵消了对NPo的影响，这表明与孔形成α亚基的额外相互作用。他莫昔芬与孔的相互作用得到了四乙基铵的浓度-响应曲线右移的支持;类似的结果是明显的与伊比利亚毒素和charybdotoxin块。我们的数据表明，溴乙烷他莫昔芬不容易穿越质膜，他莫昔芬的结合负责BK通道的激活是在细胞外的网站。他莫昔芬结合位点可以在BK通道β 1亚基的细胞外环内，或者在具有细胞外结合位点的尚未鉴定的介体上。
Smooth-muscle calcium-activated large-conductance potassium channels (BK channels) are activated by tamoxifen and 17-beta-estradiol. This increase in NPo, the number of channels, N, multiplied by open probability, depends on the presence of the regulatory beta1-subunit. Furthermore, a previous study indicated that 17-beta-estradiol might bind an extracellular site on the beta1-subunit. Because tamoxifen and 17-beta-estradiol may share a common binding site, we hypothesized that tamoxifen activates BK channels through a site on the extracellular surface of the membrane. A membrane-impermeant analog of tamoxifen, ethylbromide tamoxifen, was synthesized and used to test this hypothesis in whole-cell, outside-out, cell-attached, and inside-out patches from canine colonic smooth muscle cells. Ethylbromide tamoxifen is positively charged and is therefore membrane-impermeant. In whole-cell experiments, ethylbromide tamoxifen increased K+ current at potentials positive to +40 mV, which has previously been attributed to BK channels. Unlike tamoxifen, ethylbromide tamoxifen did not inhibit delayed rectifier current. In outside-out patches, ethylbromide tamoxifen increased BK channel NPo with an EC50 value of 1 muM. Ethylbromide tamoxifen did not increase BK channel NPo in cell-attached or inside-out patches; however, subsequent addition of equimolar tamoxifen did. Both drugs diminished BK channel unitary conductance to a degree that paralleled the effect on NPo, suggesting an additional interaction with the pore-forming alpha-subunit. An interaction of tamoxifen with the pore was supported by a right shift in the concentration-response curve for tetraethylammonium; similar results were evident with iberiotoxin and charybdotoxin block. Our data suggest that ethylbromide tamoxifen does not easily traverse the plasma membrane and that tamoxifen binding responsible for activation of BK channels is at an extracellular site. The tamoxifen binding site may be within the extracellular loop of the BK channel beta1-subunit or, alternatively, on an as-yet-unidentified mediator that has an extracellular binding site.