Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma.
Cholesterol activates BK channels by increasing KCNMB1 protein levels in the plasmalemma.
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DOI：
10.1016/j.jbc.2021.100381
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Dopico AM
中科院分区：
文献类型：
--
作者：
Bukiya AN;Leo MD;Jaggar JH;Dopico AM
钙/电压门控的大电导钾通道（BK通道）控制着关键的生理过程，包括平滑肌收缩。大量观察结果一致表明，膜胆固醇（CLR）升高会抑制由形成通道的α亚基组成的同型BK通道的活性。然而，在哺乳动物平滑肌中，天然的BK通道包含辅助性的KCNMB1（β1）亚基，这使得BK通道在生理细胞内钙浓度下能够被激活。在此，我们研究了CLR富集对大鼠大脑动脉肌细胞BK电流的影响。在游离钙离子浓度[Ca²⁺]为30 μM的条件下，利用大脑中动脉（MCA）肌细胞的内面向外式膜片，我们检测到肌细胞在体内和体外CLR富集时BK通道的激活情况。虽然在体外加载CLR的5分钟内肌细胞的CLR显著增加，但这种对膜片的短暂CLR富集却降低了BK电流，这表明CLR对BK通道的激活需要一个持续的细胞过程。事实上，用布雷菲德菌素A（BFA）阻断细胞内蛋白质运输，不仅阻止了BK通道的激活，而且在CLR富集时导致通道受到抑制。表面蛋白质生物素化结合蛋白质印迹法显示，BFA阻断了通过CLR富集实现的质膜KCNMB1水平的升高。此外，对具有天然高KCNMB1水平的动脉（如基底动脉和冠状动脉）进行CLR富集未能激活BK电流。最后，在KCNMB1⁻/⁻小鼠的MCA肌细胞中，CLR富集未能激活BK通道，而在其野生型（C57BL/6）对应细胞中则检测到激活。总之，BK通道的CLR调节从抑制到激活的转变是由KCNMB1亚基膜水平的依赖于运输的升高所决定的。
Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.
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影响因子：
5.5
作者：
Gu, Ruo-Xu;Baoukina, Svetlana;Tieleman, D. Peter
通讯作者：
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DOI：
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发表时间：
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影响因子：
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DOI：
10.1085/jgp.116.3.411
发表时间：
2000-09
期刊：
The Journal of general physiology
影响因子：
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Cox DH;Aldrich RW
通讯作者：
Aldrich RW