Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells
Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells
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DOI：
10.1152/ajpcell.00364.2002
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发表时间：
2003-03-01
影响因子：
5.5
通讯作者：
Singer, JJ
中科院分区：
文献类型：
--
作者：
Clarke, AL;Petrou, S;Singer, JJ
脂肪酸和其他带负电荷的单链脂质增加大电导Ca 2+激活的K+（BKCa）通道活性，而鞘氨醇和其他带正电荷的单链脂质抑制活性。因为这些分子对由内而外和由外而外的斑块都有效，而且因为它们可以在双层中翻转，所以它们的作用位点的位置还不清楚。为了确定带电脂质在该通道上的作用位点，我们使用了两种不太可能翻转脂质双层的化合物。棕榈酰辅酶A（PCoA）用于鉴定带负电荷的脂质的作用位点，而带正电荷的肉豆蔻酰化五肽（myr-KPRPK）用于研究带正电荷的脂质的作用位点。使用膜片钳技术在切除的膜片中研究了这些化合物对通道活性的影响。在“正常”离子强度溶液和高离子强度溶液用于屏蔽膜表面电荷的实验中，PCoA仅在应用于外向贴片时增加通道活性，表明带负电荷的脂质的作用部位位于膜的外表面。类似于其他带正电荷的脂质的活性降低，仅当myr-KPRPK应用于外向型贴片时观察到，这表明带正电荷的脂质通过也作用于外膜表面来抑制活性。还描述了myr-KPRPK和KPRPK的一些通道阻断作用。作用的侧性表明，单链脂质对通道活性的调节可以通过与通道蛋白的相互作用而发生。
Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca2+-activated K+ (BKCa) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In "normal" ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.