Department of Pharmacology, Addiction Science, and Toxicology, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA.
Department of Physiology, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee, USA.
J Biol Chem. 2021 Jan-Jun;296:100381. doi: 10.1016/j.jbc.2021.100381. Epub 2021 Feb 6.
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 (β) 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 [Ca]=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.
钙/电压门控、大电导钾通道（BK）控制着包括平滑肌收缩在内的关键生理过程。大量观察结果表明，升高的膜胆固醇（CLR）会抑制由通道形成的α亚基组成的同型 BK 的活性。然而，在哺乳动物平滑肌中，天然的 BK 包括辅助性 KCNMB1（β）亚基，使 BK 在生理细胞内钙水平下激活。在这里，我们研究了 CLR 富集对大鼠脑动脉心肌细胞 BK 电流的影响。在 [Ca]=30 μM 时，使用来自大脑中动脉（MCA）心肌细胞的内面向外膜片，我们检测到了对体内和体外 CLR 富集的心肌细胞的 BK 激活。虽然在 5 分钟内实现了心肌细胞 CLR 的显著增加，但细胞膜片的这种短暂的 CLR 富集降低了 BK 电流，表明 CLR 对 BK 的激活需要一个长期的细胞过程。事实上，用布雷菲德菌素 A（BFA）阻断细胞内蛋白运输不仅阻止了 BK 的激活，而且在 CLR 富集时导致通道抑制。随后的表面蛋白生物素化和 Western blot 显示，BFA 阻止了通过 CLR 富集实现的质膜 KCNMB1 水平的增加。此外，如基底动脉和冠状动脉等自然具有高 KCNMB1 水平的动脉中的 CLR 富集未能激活 BK 电流。最后，CLR 富集未能激活 KCNMB1 基因敲除小鼠的 MCA 心肌细胞中的 BK 通道，而在其野生型（C57BL/6）对照中则检测到激活。总之，CLR 对 BK 的调节从抑制到激活的转变是由膜 KCNMB1 亚基的转运依赖性增加决定的。
