Monitoring Receptor-Mediated Changes of Intracellular cAMP Level by Using Ion Channels and Fluorescent Proteins as Biosensors

Excerpt

Five-hydroxytryptamine (5-HT or serotonin) is an important neuromodulator involved in a wide range of physiological functions. The effects of serotonin are mediated by a large family of receptors, either ionotropic or coupled to second-messenger cascades. With the exception of the 5-HT₃ receptor, which is a cation channel, all 5-HT receptors belong to the superfamily of 7 transmembrane-spanning receptors that are coupled to multiple heterotrimeric G-proteins.

Many of the cellular responses mediated by serotonin do not involve activation of one particular second-messenger cascade but result from the functional integration of the networks of intracellular signaling pathways. To better understand serotonergic signaling, it is therefore important to have a broad palette of methodical approaches that allow specific analysis of signaling processes with high spatial and temporal resolution. Moreover, study of receptor functions within a living cell is required to extend results obtained by biochemical and pharmacological methods. Such measurements also allow real-time analysis of signaling processes in a single cell.

Cyclic AMP (cAMP) is a key second messenger that transmits information to many different effector proteins within the cell. The cellular cAMP level depends on the activity of two groups of enzymes, the adenylyl cyclases (AC) that produce cAMP and the phosphodiesterases (PDE) that hydrolyze cAMP (Beavo, 1995; Sunahara et al., 1996). Increased cAMP levels activate a number of different effector proteins, including protein kinase A (PKA) (Francis and Corbin, 1999), hyperpolarization-activated (I_h) channels (DiFrancesco, 1993), the guanine–nucleotide exchange factor Epac (de Rooij et al., 1998), and cyclic nucleotide-gated (CNG) channels (Finn et al., 1996). Metabotropic serotonin receptors coupled to Gs (5HT4 and 5HT7) or Gi/o proteins (5HT1 and 5HT5) regulate AC activity, thereby changing local cAMP concentration (Barnes and Sharp, 1999).

Because biochemical methods used for cAMP measurement lack both spatial and temporal resolution, detailed understanding of how information is transduced within cAMP-regulated signaling cascades is elusive. The classical approach to analyze the receptor-mediated change in cAMP concentration includes labeling of the cells with radioactive adenine followed by the calculation of the conversion rate of [³H]ATP to [³H]cAMP. Although this biochemical assay is very robust and reproducible, it can not provide information about the real-time course of the cAMP level. To answer this question, cAMP signals need to be measured within the dynamic environment of the living cell.

In this chapter, we concentrate on recently established methods allowing the quantitative measurement of the intracellular cAMP concentration in living cells with good spatial and temporal resolution. These will include two different approaches: (1) electrophysiological analysis to detect electrical currents mediated by cAMP-mediated activation/inactivation of hyperpolarization-activated, cyclic nucleotide-modulated ion channels (HCN) and (2) measurement of Förster Resonance Energy Transfer (FRET) by using fluorescent-labeled guanine nucleotide exchange factor (Epac) that is activated by direct binding of cAMP (Ponsioen et al., 2004).