Mirotznik R R, Zheng X, Stanley E F
Synaptic Mechanisms Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Neurosci. 2000 Oct 15;20(20):7614-21. doi: 10.1523/JNEUROSCI.20-20-07614.2000.
The inhibition of presynaptic calcium channels via G-protein-dependent second messenger pathways is a key mechanism of transmitter release modulation. We used the calyx-type nerve terminal of the chick ciliary ganglion to examine which G-proteins are involved in the voltage-sensitive inhibition of presynaptic N-type calcium channels. Adenosine caused a prominent inhibition of the calcium current that was totally blocked by pretreatment with pertussis toxin (PTX), consistent with an exclusive involvement of G(o)/G(i) in the G-protein pathway. Immunocytochemistry was used to localize these G-protein types to the nerve terminal and its transmitter release face. We used two approaches to test for modulation by other G-protein types. First, we treated the terminals with ligands for a variety of G-protein-linked neurotransmitter receptor types that have been associated with different G-protein families. Although small inhibitory effects were observed, these could all be eliminated by PTX, indicating that in this terminal the G(i) family is the sole transmitter-induced G-protein inhibitory pathway. Second, we examined the kinetics of calcium channel inhibition by uncaging the nonselective and irreversible G-protein activator GTPgammaS, bypassing the receptors. A large fraction of the rapid GTPgammaS-induced inhibition persisted, consistent with a G(o)/G(i)-independent pathway. Immunocytochemistry identified G(q), G(11), G(12), and G(13) as potential PTX-insensitive second messengers at this terminal. Thus, our results suggest that whereas neurotransmitter-mediated calcium channel inhibition is mainly, and possibly exclusively, via G(o)/G(i), other rapid PTX-insensitive G-protein pathways exist that may involve novel, and perhaps transmitter-independent, activating mechanisms.
通过G蛋白依赖性第二信使途径对突触前钙通道的抑制是递质释放调节的关键机制。我们利用鸡睫状神经节的花萼型神经末梢来研究哪些G蛋白参与突触前N型钙通道的电压敏感性抑制。腺苷引起钙电流的显著抑制，该抑制被百日咳毒素（PTX）预处理完全阻断，这与G(o)/G(i)在G蛋白途径中的唯一参与一致。免疫细胞化学用于将这些G蛋白类型定位到神经末梢及其递质释放面。我们使用两种方法来测试其他G蛋白类型的调节作用。首先，我们用与不同G蛋白家族相关的多种G蛋白偶联神经递质受体类型的配体处理神经末梢。虽然观察到了小的抑制作用，但这些作用都可以被PTX消除，表明在这个神经末梢中，G(i)家族是唯一由递质诱导的G蛋白抑制途径。其次，我们通过解开非选择性和不可逆的G蛋白激活剂GTPγS来绕过受体，研究钙通道抑制的动力学。很大一部分快速的GTPγS诱导的抑制持续存在，这与一条不依赖G(o)/G(i)的途径一致。免疫细胞化学鉴定出G(q)、G(11)、G(12)和G(13)是该神经末梢潜在的对PTX不敏感的第二信使。因此，我们的结果表明，虽然神经递质介导的钙通道抑制主要且可能仅通过G(o)/G(i)，但存在其他快速的对PTX不敏感的G蛋白途径，这些途径可能涉及新的、也许与递质无关的激活机制。
