cAMP Binding Research Articles

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Comparison of cyclic AMP binding activity within HCN channel subfamily using atomistic molecular dynamics simulations of isolated cyclic nucleotide binding domains.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a subfamily of homotetrameric, voltage gated potassium/sodium channels whose activity is modulated by cyclic AMP (cAMP). The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region which is induced by the binding of cAMP is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that affects conductance. Although various static structures of the four HCN isoforms with and without bound cyclic nucleotide have been solved and deposited on the Protein Data Bank, there is still a need to understand the precise binding interactions which lead to different sensitivities and selectivities for cyclic nucleotides within the subfamily. Using atomistic molecular dynamics, we have simulated the isolated CNBD of HCN1-4 bound to cAMP and have used both equilibrium simulations and alchemical free energy perturbation to characterize and compare the binding activities of the different isoforms. These simulations highlight the differential cyclic nucleotide binding behavior between HCN channels.

Discrimination between cyclic-nucleotides by cyclic-nucleotide gated ion channels.

Cyclic-nucleotide-gated (CNG) ion channels play important roles throughout the entire nervous system, particularly in the signal transduction of the retina and olfactory system. They are regulated by the cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). These two chemically very similar molecules bind to a specialized intracellular domain called the cyclic nucleotide-binding domain (CNBD). CNG channels are key in translating the chemical signal of the second messenger molecules cAMP and cGMP into an electrical response by inducing a conformational change in the CNBD that modulates the opening of the channel gate in the pore domain allowing the flow of ions across cell membranes. Functional measurements have shown that, in SthK (a prokaryotic homolog with high sequence and structural similarities to CNG channels), cGMP barely leads to pore opening while cAMP activates the channel. Here we address the question of how the CNBD can finely discriminate between the two chemically and structurally very similar ligands. Using a combination of atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS), and force probe molecular dynamics simulations, we elucidate, quantitatively and at the atomic level, the mechanism of CNBD ligand discrimination in SthK. We find that cAMP binds to the SthK CNBD with a slightly stronger affinity than cGMP, and can access a deep-bound state that is inaccessible to cGMP. We propose that the deep binding of cAMP is the discriminatory state that is essential for cAMP-dependent channel activation. We identify crucial residues and dynamic hydrogen bonding patterns within the binding pocket that give rise to this discrimination.

Specific interactions critical for the gating of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are named appropriately given their ability to open upon hyperpolarization and are modulated by cyclic nucleotides through their cyclic nucleotide binding domain. HCN channels contain six transmembrane domains with S1-S4 forming the voltage-sensing domain and S5-S6 forming the pore domain. HCN channels are permeable to both potassium and sodium ions, playing a key role in a wide range bodily processes such as maintaining heart rate to neuropathic pain signaling. One key difference within the HCN family is, unlike mammalian HCN channels, sea urchin HCN (spHCN) channels undergo a rapid inactivation in the absence of cAMP. This inactivation process remains unclear, it has been proposed that the C-linker plays a role where binding of cAMP prevents a rotation in the A’ helix that is suggested to lead to the inactivation of the channel. Another study has highlighted a single point mutation, F459L, is able to abolish spHCN inactivation. Here we investigated both the C-linker and F459 residue as well as other sites involved in HCN channel gating using voltage clamp fluorometry and inside-out patch clamp in spHCN channels expressed in Xenopus laevis oocytes. Our results highlight some key areas across the HCN channel that may play a role in stabilizing the gate and the inactivation phenomenon seen in spHCN.

Nanobody-mediated modulation of HCN1 channels in epileptic disorders.

Hyperpolarization-activated cyclic-nucleotide gated 1 (HCN1) channels contribute to the cationic Ih current in neurons and are involved in the control of neuronal excitability. Several mutations in the HCN1 gene have been correlated with the insurgence of Early Infantile Epileptic Encephalopathy (EIEE), a severe form of epilepsy characterized by recurrent seizures with impaired cognitive and motor development with onset in the first year of life, that doesn’t have a targeted treatment yet. In search of modulators that can reduce, and possibly abolish, HCN1-related phenotypes, nanobodies (NBs) represents a future therapeutic tool with great potential. By screening a synthetic library, we have isolated several nanobodies that specifically target HCN channel subtypes. We show here that nanobody #5 is able to revert the phenotype, 8 mV right shift in voltage-dependency, caused by the pathogenic R548H mutation of the hHCN1 channel. Notably, the nanobody acts from the extracellular side of the channel presumably binding to the voltage sensor, while the mutation, previously characterized, affects cAMP binding to the intracellular Cyclic Nucleotide Binding Domain (CNBD) of the channel. This result opens valuable therapeutical perspectives for EIEE patients and further highlights that in HCN channels voltage sensing at the membrane and ligand binding at the cytosolic domain are mutually interconnected.

Membrane phospholipids induce moderate positive cooperativity in closed pacemaker channels.

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) play pivotal role in pacemaking activity in the heart and brain. Previous studies have shown the effects of phospholipids like PIP2 on voltage dependent activation of HCN channels. Similar lipid dependent effects on channel activity have also been reported for other ion channels at an ensemble level. However, there is a lack of information regarding how ligand binding to membrane proteins itself is affected by membrane phospholipids at a molecular level. Here, we probe cAMP binding to full length HCN1 channel reconstituted into nanodiscs containing soy polar lipids to mimic a membrane environment. HCN1 nanodiscs were immobilized on zero mode waveguides (ZMWs) and binding of fluorescently labelled cAMP (fcAMP) was monitored over time using total-internal-reflection-fluorescence microscopy (TIRF). Surprisingly, we observed a moderate positive cooperativity in fcAMP binding to the closed HCN1 channel. The observed bound probability distributions deviated from the expected binomial distribution from an identical and independent binding model. These results are significantly different from the cAMP binding to the closed HCN1 channel in detergent micelles.2 Our results shed new light on the contribution of membrane phospholipids to cooperativity in ligand binding to HCN channels.

HCN2 Ion Channels Drive Pain in Rodent Models of Migraine.

Migraine is believed to be initiated by neuronal activity in the CNS, that triggers excitation of nociceptive trigeminal ganglion (TG) nerve fibers innervating the meninges and thus causes a unilateral throbbing headache. Drugs that precipitate or potentiate migraine are known to elevate intracellular levels of the cyclic nucleotides cAMP or cGMP, while anti-migraine treatments couple to signaling pathways that reduce cAMP or cGMP, suggesting an involvement of these cyclic nucleotides in migraine. Members of the HCN ion channel family are activated by direct binding of cAMP or cGMP, suggesting in turn that a member of this family may be a critical trigger of migraine. Here, we show that pharmacological block or targeted genetic deletion of HCN2 abolishes migraine-like pain in three rodent migraine models (in both sexes). Induction of migraine-like pain in these models triggered expression of the protein C-FOS, a marker of neuronal activity, in neurons of the trigeminocervical complex (TCC), where TG neurons terminate, and C-FOS expression was reversed by peripheral HCN2 inhibition. HCN2 block in vivo inhibited both evoked and spontaneous neuronal activity in nociceptive TG neurons. The NO donor glyceryl trinitrate (GTN) caused an increase in cGMP in the TG in vivo Exposing isolated TG neurons to GTN caused a rightward shift in the voltage dependence of HCN currents and thus increased neuronal excitability. This work identifies HCN2 as a novel target for the development of migraine treatments.SIGNIFICANCE STATEMENT Migraine is believed to be initiated by localized excitability of neurons within the CNS, but the most disturbing symptom, the characteristic throbbing migraine headache pain, is widely agreed to be caused by activity in afferent pain-sensitive (nociceptive) nerve fibers of the trigeminal nerve. Using a variety of preclinical models of migraine, we identify the HCN2 ion channel as the molecular source of trigeminal hyperexcitability in migraine and we show that pharmacological or genetic inhibition of HCN2 can relieve migraine-like pain symptoms. The work highlights the HCN2 ion channel as a potential pharmacological target for the development of novel analgesics effective in migraine.

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A′-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A′-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A′-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

Mechanistic Insight into the Enzymatic Inhibition of β-Amyrin against Mycobacterial Rv1636: In Silico and In Vitro Approaches.

Simple SummaryRv1636 is a mycobacterial universal stress protein whose expression level increases in different type of stress conditions. This protein promotes the growth of Mycobacterium tuberculosis in the host derived stress conditions generated during infection. Therefore in this manuscipt, we are trying to target Rv1636 using natural inhibitor. Targeting essential Mycobacterial protein using natural prodect was hypothesized to generate a molecule with low toxic effects and high inhibitory activity. It was found that Rv1636 contains ATPase activity and its ATPase activity gets disturbed by addition of β-Amyrin in the reaction. β-Amyrin was forund to interfere with the ATP binding site of Rv1636 which was confirmed by molecular docking anad dynamic studies. In addition to the ATPase activity, Rv1636 was also contain the cAMP binding capacity and also involved in balancing the cAMP levels inside cells. So, targeting Rv1636 using β-Amyrin disrupts its ATPase activity and cAMP regulatory activity and these conditions might make Mycobacterium tuberculosis more susceptible to the host derived stress conditions.Mycobacterium tuberculosis has seen tremendous success as it has developed defenses to reside in host alveoli despite various host-related stress circumstances. Rv1636 is a universal stress protein contributing to mycobacterial survival in different host-derived stress conditions. Both ATP and cAMP can be bound with the Rv1636, and their binding actions are independent of one another. β-Amyrin, a triterpenoid compound, is abundant in medicinal plants and has many pharmacological properties and broad therapeutic potential. The current study uses biochemical, biophysical, and computational methods to define the binding of Rv1636 with β-Amyrin. A substantial interaction between β-Amyrin and Rv1636 was discovered by molecular docking studies, which helped decipher the critical residues involved in the binding process. VAL60 is a crucial residue found in the complexes of both Rv1636_β-Amyrin and Rv1636-ATP. Additionally, the Rv1636_β-Amyrin complex was shown to be stable by molecular dynamics simulation studies (MD), with minimal changes observed during the simulation. In silico observations were further complemented by in vitro assays. Successful cloning, expression, and purification of Rv1636 were accomplished using Ni-NTA affinity chromatography. The results of the ATPase activity assay indicated that Rv1636’s ATPase activity was inhibited in the presence of various β-Amyrin concentrations. Additionally, circular dichroism spectroscopy (CD) was used to examine modifications to Rv1636 secondary structure upon binding of β-Amyrin. Finally, isothermal titration calorimetry (ITC) advocated spontaneous binding of β-Amyrin with Rv1636 elucidating the thermodynamics of the Rv1636_β-Amyrin complex. Thus, the study establishes that β-Amyrin binds to Rv1636 with a significant affinity forming a stable complex and inhibiting its ATPase activity. The present study suggests that β-Amyrin might affect the functioning of Rv1636, which makes the bacterium vulnerable to different stress conditions.

STK25 inhibits PKA signaling by phosphorylating PRKAR1A.

SUMMARYIn the heart, protein kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta-adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits that inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of type II regulatory subunits promotes PKA activation; however, the role of phosphorylation in type I regulatory subunits remain uncertain. Here, we utilize human induced pluripotent stem cell cardiomyocytes (iPSC-CMs) to identify STK25 as a kinase of the type Iα regulatory subunit PRKAR1A. Phosphorylation of PRKAR1A leads to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminishes Prkar1a phosphorylation, increases Pka activity, and augments contractile response to beta-adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A.

Regulation of HCN Channels by Protein Interactions.

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels are key regulators of subthreshold membrane potentials in excitable cells. The four mammalian HCN channel isoforms, HCN1-HCN4, are expressed throughout the body, where they contribute to diverse physiological processes including cardiac pacemaking, sleep-wakefulness cycles, memory, and somatic sensation. While all HCN channel isoforms produce currents when expressed by themselves, an emerging list of interacting proteins shape HCN channel excitability to influence the physiologically relevant output. The best studied of these regulatory proteins is the auxiliary subunit, TRIP8b, which binds to multiple sites in the C-terminus of the HCN channels to regulate expression and disrupt cAMP binding to fine-tune neuronal HCN channel excitability. Less is known about the mechanisms of action of other HCN channel interaction partners like filamin A, Src tyrosine kinase, and MinK-related peptides, which have a range of effects on HCN channel gating and expression. More recently, the inositol trisphosphate receptor-associated cGMP-kinase substrates IRAG1 and LRMP (also known as IRAG2), were discovered as specific regulators of the HCN4 isoform. This review summarizes the known protein interaction partners of HCN channels and their mechanisms of action and identifies gaps in our knowledge.

Computational, biochemical and ex vivo evaluation of xanthine derivatives against phosphodiesterases to enhance the sperm motility

Enhancing sperm motility in vitro has immensely benefited assisted conception methods. Phosphodiesterases (PDE) break the second messenger cAMP, and therefore, inhibition of their catalytic activity enhances the sperm motility through maintaining cAMP homeostasis in sperm. In view of identifying the molecules that could inhibit PDE functioning in spermatozoa, we aimed to evaluate the phosphodiesterase inhibitors (PDEI) - xanthine derivatives - acefylline, dyphylline and proxyphylline to repurpose them for assisted reproductive technology. These are available in the market as pharmaceutical agents to treat mainly respiratory system diseases. Based on the structure guided in silico studies, we predicted that these molecules bind to the cAMP binding catalytic pocket of PDE enzymes, and further molecular dynamics simulation analysis indicated that these molecules form the stable complexes. Isothermal titration calorimetry studies revealed that acefylline has better affinity towards PDE4A, PDE4D and PDE10A, when compared to dyphylline and proxyphylline. In addition, ex vivo studies corroborated in vitro binding studies that acefylline has much superior sperm motility enhancement property on human ejaculated spermatozoa and mouse testicular spermatozoa compared to dyphylline and proxyphylline. Communicated by Ramaswamy H. Sarma

BS-400-08 HETEROGENOUS CAMP SIGNALING IN THE INTACT HEART IS SEX DEPENDENT

cAMP is key for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. Yet, if cAMP signaling occurs heterogeneously throughout the intact heart, and how this translates into functional responses, has not been explored. To determine the spatiotemporal kinetics of cAMP signaling in the intact heart and the underlying mechanisms responsible. Male and female cardiac-specific CAMPER reporter mice that report cAMP binding by changes in FRET were used at 12 weeks. Hearts were excised and Langendorff-perfused for simultaneous cAMP and Vm imaging on a novel integrated whole heart optical imaging system. Following perfusion, tissue samples from base and apex were assessed for phosphodiesterase (PDE) activity. In male hearts, cAMP was uniformly activated in response to β-AR stimulation with bolus norepinephrine (NE, 1.5 μM). Conversely, in female hearts NE led to a greater change in cAMP activity in basal regions vs. the apex (n=7, p<0.05). Moreover, cAMP deactivation was slower in the base vs. apex, in female (n=6, p<0.01) but not male hearts (n=6). Apex-base differences were also evident following PDE inhibition with IBMX (100 μM), with a greater change in cAMP activity in the apex vs. base in both female (n=7, p<0.001) and male hearts (n=5, p<0.05). Likewise, PDE activity assays showed higher total PDE activity in apical regions (n=10, p<0.01), with more apical PDE activity in female vs. male hearts (n=5, p<0.05). In female hearts, faster apical cAMP deactivation following bolus NE was associated with a significant difference in action potential duration (APD80) between apex and base (n=3, p<0.05), but APD80 was not significantly different between regions in male hearts. Using novel whole heart imaging, we have shown female hearts display lower maximal cAMP activity and faster deactivation in the apex, in part, due to elevated PDE activity in this region. This heterogeneity was not observed in male hearts. These findings may have important implications for electrophysiological responses regulated by the cAMP pathway, particularly in heart failure, where PDE activity is altered.

Non‐canonical Recruitment of PKA Catalytic Subunits to RIα‐driven Biomolecular Condensates

A regulatory subunit of cAMP‐dependent protein kinase (PKA), RIα, was recently shown to undergo liquid‐liquid phase separation (LLPS), which is critical for cAMP compartmentation. These RIα biomolecular condensates form upon cAMP production, contain high levels of cAMP and active PKA catalytic (PKAcat) subunits, buffer increases in cAMP concentration, and suppress tumorigenic phenotypes. Through mutagenesis studies of key residues within different domains of RIα, we found that cAMP binding to both CNB‐A and B domains is required to stimulate RIα LLPS and that the dimerization via both the dimerization/docking (D/D) domain and the N3A motif of the CNB‐A is required for RIα LLPS. Our data suggests that the inhibitory sequence (IS), which binds and inhibits the catalytic subunit, is also involved in the regulation of RIα LLPS as well as recruitment of the catalytic subunit to the RIα puncta, likely through a non‐canonical interaction. RIα mutants carrying point mutations (G98S and A99S) in the IS showed enhanced RIα LLPS but weakened R:C complex and decreased capability to recruit PKAcat to RIα puncta. Intriguingly, these mutants generated puncta that did not disassemble when cAMP levels decreased, whereas puncta from wildtype RIα showed complete disassembly, suggesting that the IS motif impacts the dynamics of RIα LLPS. Our study led to a further understanding of the stimulation, formation, PKAcat recruitment, and disassembly of PKA biomolecular condensates, which may lead to novel therapeutic approaches for diseases driven by a loss of PKA RIα LLPS, such as fibrolamellar carcinoma.

Novel Hyperspectral imaging approaches allow 3D measurement of cAMP signals in localized subcellular domains of human airway smooth muscle cells.

Studies of the cAMP signaling pathway have led to the hypothesis that localized cAMP signals regulate distinct cellular responses. Much of this work focused on measurement of localized cAMP signals using cAMP sensors based upon Fӧrster resonance energy transfer (FRET). FRET-based probes are comprised of a cAMP binding domain sandwiched between donor and acceptor fluorophores. Binding of cAMP triggers a conformational change which alters FRET efficiency. In order to study localized cAMP signals, investigators have targeted FRET probes to distinct subcellular domains. This approach allows detection of cAMP signals at distinct subcellular locations. However, these approaches do not measure localized cAMP signals per se, rather they measure cAMP signals at specific locations and typically averaged throughout the cell. To address these concerns, our group implemented hyperspectral imaging approaches for measuring highly multiplexed signals in cells and tissues. We have combined these approaches with custom analysis software implemented in MATLAB and Python. Images were filtered both spatially and temporally, prior to adaptive thresholding (OTSU) to detect cAMP signals. These approaches were used to interrogate the distributions of isoproterenol and prostaglandin-triggered cAMP signals in human airway smooth muscle cells (HASMCs). Results demonstrate that cAMP signals are spatially and temporally complex. We observed that isoproterenol- and prostaglandin-induced cAMP signals are triggered at the plasma membrane and in the cytosolic space. We are currently implementing analysis approaches to better quantify and visualize the complex distributions of cAMP signals. This work was supported by NIH P01HL066299, R01HL058506, and S10RR027535.

Altered cyclic nucleotide binding and pore opening in a diseased human HCN4 channel

A growing number of nonsynonymous mutations in the human HCN4 channel gene, the major component of the funny channel of the sinoatrial node, are associated with disease but how they impact channel structure and function, and, thus, how they result in disease, is not clear for any of them. Here, we study the S672R mutation, in the cyclic nucleotide-binding domain of the channel, which has been associated with an inherited bradycardia in an Italian family. This may be the best studied of all known mutations, yet the underlying molecular and atomistic mechanisms remain unclear and controversial. We combine measurements of binding by isothermal titration calorimetry to a naturally occurring tetramer of the HCN4 C-terminal region with a mathematical model to show that weaker binding of cAMP to the mutant channel contributes to a lower level of facilitation of channel opening at submicromolar ligand concentrations but that, in general, facilitation occurs over a range that is similar between the mutant and wild-type because of enhanced opening of the mutant channel when liganded. We also show that the binding affinity for cGMP, which produces the same maximum facilitation of HCN4 opening as cAMP, is weaker in the mutant HCN4 channel but that, for both wild-type and mutant, high-affinity binding of cGMP occurs in a range of concentrations below 1 μM. Thus, binding of cGMP to the HCN4 channel may be relevant normally in vivo and reduced binding of cGMP, as well as cAMP, to the mutant channel may contribute to the reduced resting heart rate observed in the affected family.

Identification of a distinct lock system for cAMP binding in HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the Ih/If current, which plays a key role in controlling rhythmic activity in pacemaker cells. HCN channels are activated by voltage and regulated by direct binding of cAMP to their cytoplasmic C-terminal Cyclic Nucleotide Binding Domain (CNBD). The Cryo-EM structure of HCN1 revealed the presence of two helices, D and E, that fold upon cAMP binding and establish a contact to the upstream CNBD. We thus investigated the hypothesis that helices D and E regulate cAMP affinity.

Selective inhibition and quantification of endogenous cAMP binding sites in supported native membranes for single-molecule studies

Single membrane proteins are often studied in solubilized or reconstituted form to avoid endogenous background signals. However, such an artificial experimental environment, generated by the required purification protocols, might alter functional properties of the proteins investigated. Here we demonstrate on the example of the HCN2 pacemaker channel in supported membranes, that the endogenous background can be reduced by a suitable inhibitor protocol. Additionally, quantitative insight into the density of endogenous cAMP binding sites in the membrane can be obtained.

2',3'-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana.

The role of the RNA degradation product 2′,3′-cyclic adenosine monophosphate (2′,3′-cAMP) is poorly understood. Recent studies have identified 2′,3′-cAMP in plant material and determined its role in stress signaling. The level of 2′,3′-cAMP increases upon wounding, in the dark, and under heat, and 2′,3′-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2′,3′-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2′,3′-cAMP treatment. We demonstrated that 2′,3′-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide–adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2′,3′-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2′,3′-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2′,3′-cAMP treatment influences the movement of processing bodies, confirming the role of 2′,3′-cAMP in the formation and motility of membraneless organelles.

Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes.

Signaling proteins are composed of conserved protein interaction domains that serve as allosteric regulatory elements of enzymatic or binding activities. The ubiquitous, structurally conserved cyclic nucleotide binding (CNB) domain is found covalently linked to proteins with diverse folds that perform multiple biological functions. Given that the structures of cAMP-bound CNB domains in different proteins are very similar, it remains a challenge to determine how this domain allosterically regulates such diverse protein functions and folds. Instead of a structural perspective, we focus our attention on the energy landscapes underlying the CNB domains and their responses to cAMP binding. We show that optical tweezers is an ideal tool to investigate how cAMP binding coupled to interdomain interactions remodel the energy landscape of the regulatory subunit of protein kinase A (PKA), which harbors two CNB domains. We mechanically manipulate and probe the unfolding and refolding behavior of the CNB domains as isolated structures or selectively as part of the PKA regulatory subunit, and in the presence and absence of cAMP. Optical tweezers allows us to dissect the changes in the energy landscape associated with cAMP binding, and to examine the allosteric interdomain interactions triggered by the cyclic nucleotide. This single molecule approach can be used to study other modular, multidomain signaling proteins found in nature.