Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion.
Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion.
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DOI：
10.1085/jgp.116.2.147
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发表时间：
2000-08
期刊：
影响因子：
--
通讯作者：
Karpen JW
中科院分区：
文献类型：
--
作者：
Rich TC;Fagan KA;Nakata H;Schaack J;Cooper DM;Karpen JW
环 AMP 是一种普遍存在的第二信使，可协调多种细胞功能。目前测量 cAMP 的方法缺乏时间和空间分辨率，导致人们普遍认为，与 Ca2+ 不同，cAMP 信号很简单且包含很少的信息。在这里，我们展示了腺病毒表达的环核苷酸门控通道作为 cAMP 传感器的发展。由嗅觉 α 亚基组成的同多聚体通道对 cAMP 浓度的跳跃快速响应，并且测量它们的 cAMP 灵敏度以校准用于细胞内测量的传感器。我们使用这些通道来检测单细胞和细胞群中异源表达或内源腺苷酸环化酶产生的 cAMP。毛喉素刺激后，C6-2B 神经胶质瘤细胞中的内源性腺苷酸环化酶在通道附近产生高浓度的 cAMP，但总体 cAMP 浓度仍然较低。我们发现，在全细胞膜片钳实验中大量细胞质的快速交换并不能阻止表达外源腺苷酸环化酶的人胚肾293 (HEK-293)细胞通道附近cAMP的显着水平的积累。这些结果可以通过细胞区室模型进行定量解释，其中环核苷酸门控通道与微域中的腺苷酸环化酶共定位，并且这些域和大量胞质溶胶之间的 cAMP 扩散受到显着阻碍。与模型一致，我们测量了 cAMP 从全细胞贴片移液管到通道的缓慢扩散速率（194 秒内交换了 90%，而监测与细胞质交换的物质则需要 22-56 秒）。如果没有微域和限制扩散进入细胞质，我们无法解释所有结果。值得注意的是，在无限制扩散模型中，即使在非常接近腺苷酸环化酶的情况下，cAMP 也不会达到足够高的浓度以充分激活 PKA 或环核苷酸门控通道，除非整个细胞充满 cAMP。因此，微结构域应促进 PKA 和环核苷酸门控通道的快速有效激活，并允许腺苷酸环化酶的局部反馈控制。局部 cAMP 信号还应促进细胞靶标的差异调节。
Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca2+, cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide–gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory α subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide–gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22–56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide–gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide–gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.
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影响因子：
3.4
作者：
Chen, CH;Nakamura, T;Koutalos, Y
通讯作者：
Koutalos, Y
影响因子：
4.8
作者：
Dessauer, CW;Gilman, AG
通讯作者：
Gilman, AG
影响因子：
64.8
作者：
Finch, EA;Augustine, GJ
通讯作者：
Augustine, GJ
DOI：
10.1073/pnas.95.13.7693
发表时间：
1998-06-23
影响因子：
11.1
作者：
Allen, ML;Koh, DS;Tempel, BL
通讯作者：
Tempel, BL
影响因子：
4.8
作者：
Cassano, S;Di Lieto, A;Avvedimento, EV
通讯作者：
Avvedimento, EV