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Ion channels are membrane proteins central to the generation of electrical signals throughout the body1. Unique

among the voltage-dependent channel family, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open in response to plasma membrane hyperpolarizing (negative) voltages, where most are activated by depolarizing voltage2. Upon opening, these channels give rise to a nonselective cation ‘funny’ current (If), which generates spontaneous rhythmic firing of action potential in the heart and nervous system. In the human body, one of the mechanisms by which autonomic control over heart rate is achieved is the regulation of intracellular levels of cyclic nucleotides (cNMPs) in the sinoatrial node. Increased levels of cNMPs facilitate their binding to the C-terminal cyclic nucleotide-binding domain (CNBD)3 of HCN4 channels and enhance HCN channel opening in response to voltage, resulting in increased heart rate. Previous studies have shown that cNMP-induced conformational changes in the CNBD are conferred to the pore through the C-linker domains, but the exact molecular mechanism of this transduction remains unclear. Now, Lolicato et al.4 identified a second cGMP binding site in the C-linker of HCN4 and unexpectedly uncovered cyclic dinucleotides (CDNs), a well-established group of important second messengers in bacteria and other synthetic molecules, as effective allosteric inhibitors of HCN4 modulation by cNMPs.

Since 1987, CDNs have emerged as important bacterial second messengers, regulating a variety of cellular processes5. However, these molecules have recently been shown to participate in mammalian signaling pathways as well. Activating an innate immune cell response, pathogen-derived CDNs were shown to bind the intracellular stimulator of interferon genes (STING) receptor and initiate a signaling cascade resulting in induction of translation of interferons and cytokines6. Moreover, endogenous mammalian CDN synthesis was recently discovered. After exposure to foreign nucleic acids in their cytosol, the cyclic GMP-AMP synthase of mammals was

shown to activate and catalyze the synthesis of a high-affinity ligand for STING7. With ongoing investigations on the roles bacterial and mammalian CDNs have in cellular responses to threats, the existence of other mammalian receptors, unrelated to host defense, was never explored.

In their work, Lolicato et al.4 combined structural, biochemical and functional approaches to show that CDNs are potent and specific inhibitors of HCN4 modulation by cNMPs. This implies that, for the first time, CDN signaling in mammalian cells is shown to reach outside the scope of the immune system response. Initially, the authors aimed to determine the molecular basis of the low-affinity modulation of If by cGMP, which resulted in a structure-driven discovery of an additional, ‘noncanonical’ binding site for cGMP in the C-terminal fragment of HCN4. This binding site was termed the ‘C-linker pocket’ (CLP) and is located at the interface between the C-linker and the CNBD. However, with cGMP binding effects attributed only to the well-studied

CNBD3, the attempts to dissect the functional significance of the CLP occupancy have led to creative experiments (Fig. 1).

Analysis of the dimensions of CLP showed its ability to accommodate a molecule twice the size of cGMP. Using in silico docking methods, the CDN cyclic di-(3′,5′)-guanosine monophosphate (c-di-GMP), which is important in signaling by Gram-negative bacteria, was found to be a perfect fit. Amazingly, when tested on sinoatrial node cardiomyocytes, the binding of c-di-GMP and other CDN types to HCN4 channels resulted in complete inhibition of the cAMP-induced voltage-dependent shift, mimicking the endogenous muscarinic inhibition of the If current and heart rate. This inhibition was specific to the CLP of HCN4 as CLP mutants or other channel subtypes did not respond to CDNs. The authors used computational screens of synthetic low-molecular-weight compounds to find other ligands that could occupy the CLP of HCN4. Biphenyl urea stood out from other hits found in these screens, with its inhibitory effects on

STRUCTURAL BIOLOGY

A ‘funny’ cyclic dinucleotide receptorDeciphering the molecular basis of HCN channel regulation by cGMP leads to the serendipitous discovery of cyclic dinucleotides as potent inhibitors of If current in the heart.

Yoni Haitin

Figure 1 | HCN4 channels expressed in the sinoatrial node generate the initial phase of the cardiacpacemaker action potentials. HCN4 is composed of four identical transmembrane subunits (one highlighted as a colored cartoon and the other three in surface representation; adopted from the structure with Protein Data Bank (PDB) code 4JTC). HCN4 activity is enhanced by cNMP binding at the intracellular CNBD (blue; adopted from the structure with PDB code 1Q3E). CDNs binding the CLP (asterisk) inhibit the C-linker (red) from transducing the cNMP binding signal to the gate of the channel, resulting in the reversal of cNMP-induced heart rate stimulation.

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414 nature chemical biology | VOL 10 | JUNE 2014 | www.nature.com/naturechemicalbiology

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HCN4 modulation by cAMP almost identical to those observed for the CDNs.

The serendipity of the structure-driven discovery of the modulation of HCN4 channels by CDNs, followed by screening for synthetic compounds that bind the CLP, shown in the work of Lolicato et al.4, stresses the combined effectiveness of classical and reverse pharmacology. If not for a functionally undiscerned occupancy of the CLP by cGMP, the role of CDNs in HCN4 modulation may have remained obscure.

This new cross-kingdom interplay between bacterial second messenger system and mammalian targets raises some interesting questions. From a drug discovery point of view, the markedly straightforward inhibition of HCN4 channel modulation by cNMP,

through binding of bacterial CDNs to the CLP, may imply that other, yet-to-be-explored interfaces between bacterial and mammalian systems exist. Could other drug leads exist in the microbial small-molecule milieu outside the realm of antibiotics? Focusing on basic cardiac physiology, are the newly discovered endogenous mammalian CDNs8 synthesized in heart cells? And if so, do they occur in sufficient intracellular concentrations to allow for their participation in heart rate control? Finally, do CDNs have roles in other cellular processes? With their physiological relevance to mammalian cell signaling still being unraveled, future studies on CDN regulation of additional proteins may provide clues for new, medically relevant drug targets, as in the case of HCN4. ■

Yoni Haitin is in the Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, Washington. e-mail: haitinyo@uw.edu

References1. Hille, B. Ion Channels of Excitable Membranes (Sinauer

Associates, Sunderland, MA, 2001). 2. Craven, K.B. & Zagotta, W.N. Annu. Rev. Physiol. 68,

375–401 (2006). 3. Zagotta, W.N. et al. Nature 425, 200–205 (2003). 4. Lolicato, M. et al. Nat. Chem. Biol. 10, 457–462 (2014).5. Römling, U., Galperin, M.Y. & Gomelsky, M. Microbiol. Mol.

Biol. Rev. 77, 1–52 (2013). 6. Danilchanka, O. & Mekalanos, J.J. Cell 154, 962–970

(2013). 7. Li, X. et al. Immunity 39, 1019–1031 (2013). 8. Wu, J. et al. Science 339, 826–830 (2013).

Competing financial interestsThe author declares no competing financial interests.

PLANT SIGNALING

Abscisic acid receptor hole-in-oneThe phytohormone abscisic acid (ABA) has a central role in adaptive responses of plants to abiotic stress. A new ABA antagonist offers a promising tool to study ABA signaling and adaptation to abiotic stress in diverse plants, including crops.

Ken-ichiro Hayashi & Toshinori Kinoshita

P lants are challenged by numerous environmental conditions causing biotic and abiotic stresses. ABA, a

sesquiterpene phytohormone, regulates various developmental processes such as seed dormancy, hydraulic conductivity and stomatal closure and has a central role in adaptive responses to abiotic stress such as high salt conditions, cold temperatures and drought1. Abiotic stresses elevate endogenous ABA levels and initiate a series of adaptive responses, including transcriptional activation of stress-response genes2. Because abiotic stress is known to influence plant productivity, ABA signal transduction has long been considered an attractive target to confer plant tolerance to drought and salinity2. Takeuchi et al.3 use structural insights into the ABA receptor complex to design an antagonist of ABA signaling, which may provide a useful tool for manipulating ABA responses in plants and crops.

Like other plant hormones such as gibberellins, auxins and brassinosteroids, ABA induces downstream signaling by facilitating interactions between a receptor protein and an effector protein (Fig. 1). ABA binds members of the PYR/PYL/RCAR (PYL) receptor family and thereby stabilizes a protein-protein interaction between PYL and phosphatase

type 2C proteins (PP2Cs). In doing so, ABA functions as an allosteric ligand that elicits a conformational change in the PYL receptor, which binds the active site of PP2C and inactivates PP2C activity4. ABA signaling pathways are normally repressed when ABA concentrations are low because PP2C maintains SNF1-related protein kinases in an inactive state5. The ABA–PYL–PP2C complexes that mediate ABA signaling in Arabidopsis thaliana comprise a combinatorial pairing of 14 PYL receptors and 9 members of the PP2C family6,7.

The identification of small-molecule chemical probes that specifically modulate ABA perception and signaling represents an attractive tool for plant biology. Such modulators not only can potentially overcome the PYL-PP2C receptor heterogeneity but also may reveal physiological mechanisms of ABA signaling, especially in cases where plant genetics cannot be used. ABA antagonists, in particular, have been sought after since the ABA-dependent interaction between PYL and PP2C was analyzed by crystallography8. The crystal structures revealed that ABA binding induces closure of a gating loop of PYL, which traps the ABA molecule in the binding pocket and remodels PYL to create

an interaction surface where the active site of PP2C binds.

Takeuchi et al.3 now use a structure-guided approach to design the first ABA receptor antagonist, 3′-hexylsulfanyl-ABA (AS6). An analysis of the crystal structure of the PYL–ABA–PPC2 receptor complex7 revealed that the ABA-bound PYL receptor complex forms a small hydrophobic tunnel directed toward the PP2C interaction domain. The authors hypothesized that ABA analogs would bind and induce gate closure but that adding an appendage to the molecule that would protrude into the channel could prevent PP2C binding. To test this, they synthesized a series of ABA analogs in which alkylthiol chains of increasing length were appended to the 3′ position of ABA. On the basis of modeling and binding studies, the authors focused on AS6, which shows high affinity to the ABA binding site and closes the gating loop but prevents the successive binding of PP2C to the PYL–AS6 complex. Isothermal titration calorimetry revealed that AS6 can bind PYL receptors with an affinity (Kd of 0.5–1.3 µM) similar to that of ABA (0.8 µM) in a range of PYL receptor constructs. AS6 did not affect endogenous ABA levels in plants and was found to be inert to ABA metabolic enzymes such as

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