Karpen Jeffrey W, Rich Thomas C
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239, USA.
Proc West Pharmacol Soc. 2004;47:1-5.
A large number of hormones, neurotransmitters, and odorants alter cellular behavior by triggering changes in intracellular levels of cAMP. Although the effector proteins that bind cAMP have been identified, it is not known how this one messenger can differentially regulate the activities of hundreds of cellular proteins. The spatial and temporal nature of cAMP signals and, thus, their information content remain largely unknown. We present here a high-resolution method for measuring cAMP signals near the plasma membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We show how these channels can be used in electrophysiological experiments to accurately measure changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We have found in several cell types (both excitable and nonexcitable) that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is severely hindered. We also show that a uniform extracellular stimulus can initiate very distinct cAMP signals within different compartments of a simple, nonexcitable cell. Analysis of compartmental models indicates that diffusional restrictions between microdomains (near the membrane) and the cytosol, as well as differential regulation of phosphodiesterase activity, are necessary to explain such distinct signals. Using modified CNG channels as sensors has much greater spatial and temporal resolution than other methods for measuring cAMP, and should help to unravel the complexities of signaling by this ubiquitous messenger. Cyclic AMP (cAMP), the prototypical second messenger, regulates a wide variety of cellular processes. Changes in cAMP concentration transmit information to downstream effectors including protein kinase A (PKA), cyclic nucleotide-gated (CNG) channels, hyperpolarization activated (Ih) channels, and Epac. However, it is largely unclear how differential regulation of cellular targets occurs. The concept of compartmentation emerged over 20 years ago in studies of cardiac myocytes, to help explain how a variety of extracellular stimuli that primarily act through cAMP can have very different downstream effects on the cell. The basis for compartmentation, and indeed, the nature of cAMP signals themselves, have remained mysteries. To understand how these signals function within the cell it is important to answer the following questions: (i) How are cAMP signals localized? (ii) What are the kinetics of cAMP signals in localized domains? and (iii) What information is contained in the amplitude and frequency of cAMP signals? We describe here a high-resolution method for measuring cAMP signals near the plasma membrane, using modified cyclic nucleotide-gated ion channels. This approach was inspired by the field of retinal phototransduction, the best-studied second messenger signaling system, in which elegant biochemical studies have been complemented by real-time measurements of cGMP signals using endogenous cyclic nucleotide-gated (CNG) channels. CNG channels are directly opened by the binding of cyclic nucleotides. They were discovered in retinal photoreceptor cells and olfactory receptor neurons, where they generate the electrical response to light and odorants. The native retinal channel is cGMP specific, while the native olfactory channel is equally sensitive to cAMP and cGMP. Native CNG channels consist of A and B subunits, both of which bind cyclic nucleotides, although most A subunits form functional channels on their own. We have modified an olfactory channel A subunit (CNGA2) to improve its sensitivity and selectivity for cAMP. Two of the findings with this approach, summarized here, are: (i) cAMP in several cell types is produced in subcellular compartments under the plasma membrane with restricted diffusional access to the bulk cytosol; and (ii) the amplitude and kinetics of cAMP signals within these compartments are distinct from those in the remainder of the cell.
大量的激素、神经递质和气味分子通过触发细胞内cAMP水平的变化来改变细胞行为。尽管已经鉴定出了与cAMP结合的效应蛋白，但尚不清楚这一信使如何能够差异性地调节数百种细胞蛋白的活性。cAMP信号的空间和时间特性，以及它们的信息内容在很大程度上仍然未知。我们在此介绍一种用于测量单细胞质膜附近cAMP信号的高分辨率方法。嗅觉受体神经元的环核苷酸门控（CNG）离子通道已经经过基因改造，以改善其cAMP传感特性。我们展示了这些通道如何用于电生理实验，以准确测量膜附近cAMP浓度的变化，而大多数腺苷酸环化酶都位于此处。我们在几种细胞类型（包括可兴奋细胞和非可兴奋细胞）中发现，cAMP是在质膜附近的亚细胞区室中产生的，并且cAMP从这些区室扩散到细胞质溶胶的过程受到严重阻碍。我们还表明，均匀的细胞外刺激可以在一个简单的非可兴奋细胞的不同区室中引发非常不同的cAMP信号。对区室模型的分析表明，微区（靠近膜）和细胞质溶胶之间的扩散限制以及磷酸二酯酶活性的差异调节，对于解释这种不同的信号是必要的。使用经过改造的CNG通道作为传感器，比其他测量cAMP的方法具有更高的空间和时间分辨率，并且应该有助于揭示这种普遍存在的信使的信号传导复杂性。环磷酸腺苷（cAMP）作为典型的第二信使，调节着各种各样的细胞过程。cAMP浓度的变化将信息传递给下游效应器，包括蛋白激酶A（PKA）、环核苷酸门控（CNG）通道、超极化激活（Ih）通道和交换蛋白直接激活剂（Epac）。然而，细胞靶点的差异性调节在很大程度上尚不清楚。区室化的概念在20多年前心肌细胞的研究中就已出现，以帮助解释主要通过cAMP起作用的各种细胞外刺激如何能够对细胞产生非常不同的下游效应。区室化的基础，以及实际上cAMP信号本身的性质，仍然是个谜。为了理解这些信号在细胞内如何发挥作用，回答以下问题很重要：（i）cAMP信号是如何定位的？（ii）cAMP信号在局部区域的动力学是什么？以及（iii）cAMP信号的幅度和频率包含什么信息？我们在此描述一种使用经过改造的环核苷酸门控离子通道来测量质膜附近cAMP信号的高分辨率方法。这种方法的灵感来自视网膜光转导领域，这是研究得最透彻的第二信使信号系统，在该领域中，精湛的生化研究已经通过使用内源性环核苷酸门控（CNG）通道对cGMP信号进行实时测量得到了补充。CNG通道通过环核苷酸的结合而直接打开。它们在视网膜光感受器细胞和嗅觉受体神经元中被发现，在那里它们产生对光和气味分子的电反应。天然的视网膜通道对cGMP具有特异性，而天然的嗅觉通道对cAMP和cGMP同样敏感。天然的CNG通道由A和B亚基组成，两者都结合环核苷酸，尽管大多数A亚基自身就能形成功能性通道。我们已经对一种嗅觉通道A亚基（CNGA2）进行了改造，以提高其对cAMP的敏感性和选择性。用这种方法得到的两个发现总结如下：（i）几种细胞类型中的cAMP是在质膜下的亚细胞区室中产生的，其扩散到细胞质溶胶的过程受到限制；（ii）这些区室中cAMP信号的幅度和动力学与细胞其余部分的不同。
