Liu Liwang, Rittenhouse Ann R
Program in Neuroscience, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester 01655, USA.
Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):295-300. doi: 10.1073/pnas.0136826100. Epub 2002 Dec 20.
N-type Ca(2+) channels participate in acute activity-dependent processes such as regulation of Ca(2+)-activated K(+) channels and in more prolonged events such as gene transcription and long-term depression. A slow postsynaptic M(1) muscarinic receptor-mediated modulation of N-type current in superior cervical ganglion neurons may be important in regulating these processes. This slow pathway inhibits N-type current by using a diffusible second messenger that has remained unidentified for more than a decade. Using whole-cell patch-clamp techniques, which isolate the slow pathway, we found that the muscarinic agonist oxotremorine methiodide not only inhibits currents at positive potentials but enhances N-type current at negative potentials. Enhancement was also observed in cell-attached patches. These findings provide evidence for N-type Ca(2+)-current enhancement by a classical neurotransmitter. Moreover, enhancement and inhibition of current by oxotremorine methiodide mimics modulation observed with direct application of a low concentration of arachidonic acid (AA). Although no transmitter has been reported to use AA as a second messenger to modulate any Ca(2+) current in either neuronal or nonneuronal cells, we nevertheless tested whether a fatty acid signaling cascade was involved. Blocking phospholipase C, phospholipase A(2), or AA but not AA metabolism minimized muscarinic modulation of N-type current, supporting the participation of these molecules in the slow pathway. A role for the G protein G(q) was also confirmed by blocking muscarinic modulation of Ca(2+) currents with anti-G(qalpha) antibody. Our finding that AA participates in the slow pathway strongly suggests that it may be the previously unknown diffusible second messenger.
N型钙离子通道参与急性活动依赖性过程，如对钙激活钾通道的调节，以及更持久的活动，如基因转录和长时程抑制。颈上神经节神经元中，M1毒蕈碱受体介导的对N型电流的缓慢突触后调制，可能在调节这些过程中起重要作用。这条缓慢通路通过一种尚未确定身份达十多年的可扩散第二信使来抑制N型电流。我们运用全细胞膜片钳技术分离出这条缓慢通路，发现毒蕈碱激动剂氧化震颤素甲碘化物不仅在正电位时抑制电流，在负电位时还增强N型电流。在细胞贴附式膜片中也观察到了增强现象。这些发现为经典神经递质增强N型钙电流提供了证据。此外，氧化震颤素甲碘化物对电流的增强和抑制，模拟了直接施加低浓度花生四烯酸（AA）时观察到的调制。尽管尚未有报道称任何神经递质在神经元或非神经元细胞中使用AA作为第二信使来调制任何钙电流，但我们仍测试了是否涉及脂肪酸信号级联反应。阻断磷脂酶C、磷脂酶A2或AA而非AA代谢，可使毒蕈碱对N型电流的调制最小化，支持这些分子参与缓慢通路。用抗Gqα抗体阻断毒蕈碱对钙电流的调制，也证实了G蛋白Gq的作用。我们发现AA参与缓慢通路，强烈表明它可能是之前未知的可扩散第二信使。
