Cyclic Nucleotide-regulated Ion Channels Research Articles

Fluorophore-Labeled Cyclic Nucleotides as Potent Agonists of Cyclic Nucleotide-Regulated Ion Channels.

High‐affinity fluorescent derivatives of cyclic adenosine and guanosine monophosphate are powerful tools for investigating their natural targets. Cyclic nucleotide‐regulated ion channels belong to these targets and are vital for many signal transduction processes, such as vision and olfaction. The relation of ligand binding to activation gating is still challenging, and there is a need for fluorescent probes that enable the process to be broken down to the single‐molecule level. This inspired us to prepare fluorophore‐labeled cyclic nucleotides, which are composed of a bright dye and a nucleotide derivative with a thiophenol motif at position 8 that has already been shown to enable superior binding affinity. These bioconjugates were prepared by a novel cross‐linking strategy that involves substitution of the nucleobase with a modified thiophenolate in good yield. Both fluorescent nucleotides are potent activators of different cyclic nucleotide‐regulated ion channels with respect to the natural ligand and previously reported substances. Molecular docking of the probes excluding the fluorophore reveals that the high potency can be attributed to additional hydrophobic and cation‐π interactions between the ligand and the protein. Moreover, the introduced substances have the potential to investigate related target proteins, such as cAMP‐ and cGMP‐dependent protein kinases, exchange proteins directly activated by cAMP or phosphodiesterases.

Chemical synthesis and biological activity of novel brominated 7-deazaadenosine-3',5'-cyclic monophosphate derivatives.

Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3',5'-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.

Interaction of Epac with Non-canonical Cyclic Nucleotides.

Epac1 and Epac2 are cyclic nucleotide-binding (CNB) domain containing proteins, which were originally identified as cAMP-regulated guanine nucleotide exchange factors (GEFs) for the small G-protein Rap. Therefore, Epac proteins founded next to protein kinase A (PKA) and cyclic nucleotide-regulated ion channels the third group of cAMP-responsive proteins in higher organisms. Epac proteins are involved in the regulation of several physiological processes. In particular Epac1 mediates the regulation of molecular processes underlying cell adhesion and mobility. In the pancreas activation of Epac2 potentiates the release of glucose-induced insulin secretion and received attention as a putative target for antidiabetic treatment. While the regulation of Epac by cAMP has been analysed in structural and biochemical detail, less is known on the interaction of Epac with non-canonical cyclic nucleotides. This chapter will discuss to what extent other cyclic purines than cAMP or cyclic pyrimidine could act as Epac agonists or antagonists. The focus will be on the biophysical analysis of the interaction between Epac and these cyclic nucleotides.

Recent Advances in the Discovery of Small Molecules Targeting Exchange Proteins Directly Activated by cAMP (EPAC)

3',5'-Cyclic adenosine monophosphate (cAMP) is a pivotal second messenger that regulates numerous biological processes under physiological and pathological conditions, including cancer, diabetes, heart failure, inflammation, and neurological disorders. In the past, all effects of cAMP were initially believed to be mediated by protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Since the discovery of exchange proteins directly activated by cyclic adenosine 5'-monophosphate (EPACs) in 1998, accumulating evidence has demonstrated that the net cellular effects of cAMP are also regulated by EPAC. The pursuit of the biological functions of EPAC has benefited from the development and applications of a growing number of pharmacological probes targeting EPACs. In this review, we seek to provide a concise update on recent advances in the development of chemical entities including various membrane-permeable analogues of cAMP and newly discovered EPAC-specific ligands from high throughput assays and hit-to-lead optimizations.

Pseudomonas aeruginosaExoY, a cyclic GMP- and cyclic UMP-generating nucleotidyl cyclase

Background In previous studies we showed that in addition to the cyclic nucleotides (cNMPs) cAMP and cGMP, mammalian cells also contain cCMP and cUMP [for review see [1], [2]]. cCMP and cUMP are generated by the bacterial exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis as well as nitric oxide (NO)stimulated soluble guanylyl cyclase (sGC). cCMP and cUMP activate cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and cyclic nucleotide-regulated ion channels (HCN channels). cUMP, but not cCMP, is degraded by several known phosphodiesterases (PDEs). cCMP and cUMP are also exported from cells via multidrug resistance proteins (MRPs 4 and 5). These findings suggest that cCMP and cUMP constitute second messenger molecules with distinct signalling properties. Pseudomonas aeruginosa is an important pathogenic bacterium, specifically in patients with cystic fibrosis. P. aeruginosa injects effector proteins into host cells via the type III secretion system and, thereby, manipulates their functions. One of the effector proteins of P. aeruginosa is ExoY. ExoY possesses structural similarity with CyaA and EF in the catalytic domain and was assumed to constitute an adenylyl cyclase (AC) [3]. However, the cAMP-forming capacity of ExoY is rather low. More recently, it has been shown that ExoY is capable of producing large quantities of cGMP [4]. On this background, we examined the question whether ExoY also exhibits cytidylyland uridylyl cyclase activity. Methods We transfected mammalian cells with a plasmid encoding ExoY or a catalytically inactive ExoY mutant (ExoY-M). We also infected cells with P. aeruginosa encoding ExoY or ExoY-M. In addition, we infected mice intratracheally with P. aeruginosa. We determined cNMP concentrations with highly sensitive and specific HPLC-MS/MS and HPLC-MS/TOF methods. We assessed cell morphology, apoptosis and label-free dynamic mass distribution, constituting a holistic and unbiased assay for cell activation.

A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels

Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels.

Gating of the MlotiK1 potassium channel involves large rearrangements of the cyclic nucleotide-binding domains

Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotide-regulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ∼1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.

Cyclic Nucleotide-Regulated Ion Channels: Spotlight on Symmetry

In this issue of Structure, Chiu et al. (2007) report the 16 A EM structure of the prokaryotic cyclic nucleotide-regulated K(+) channel MloK1. This structure reveals that the channel is arranged as a four-fold symmetric tetramer.

Structure and regulation of the cAMP-binding domains of Epac2.

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.