cAMP binding to closed pacemaker ion channels is cooperative.

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Pyruvate-dependent growth of Methanosarcina acetivorans.

Methanogenesis is a key step during anaerobic biomass degradation. Methanogenic archaea (methanogens) are the only organisms coupling methanogenic substrate conversion to energy conservation. The range of substrates utilized by methanogens is limited, with acetate and H2+CO2 being the ecologically most relevant. The only single methanogenic energy substrate containing more carbon-carbon bonds than acetate is pyruvate. Only the aggregate-forming, freshwater methanogen Methanosarcina barkeri Fusaro was shown to grow on this compound. Here, the pyruvate-utilizing capabilities of the single-celled, marine Methanosarcina acetivorans were addressed. Robust pyruvate-dependent, methanogenic, growth could be established by omitting CO2 from the growth medium. Growth rates which were independent of the pyruvate concentration indicated that M. acetivorans actively translocates pyruvate across the cytoplasmic membrane. When 2-bromoethanesulfonate (BES) inhibited methanogenesis to more than 99%, pyruvate-dependent growth was acetogenic and sustained. However, when methanogenesis was completely inhibited M. acetivorans did not grow on pyruvate. Analysis of metabolites showed that acetogenesis is used by BES-inhibited M. acetivorans as a sink for electrons derived from pyruvate oxidation and that other, thus far unidentified, metabolites are produced.IMPORTANCEThe known range of methanogenic growth substrates is very limited and M. acetivorans is only the second methanogenic species for which growth on pyruvate is demonstrated. Besides some commonalities, analysis of M. acetivorans highlights differences in pyruvate metabolism among Methanosarcina species. The observation that M. acetivorans probably imports pyruvate actively indicates that the capabilities for heterotrophic catabolism in methanogens may be underestimated. The mostly acetogenic growth of M. acetivorans on pyruvate with concomitant inhibition of methanogenesis confirms that energy conservation of methanogenic archaea can be independent of methane formation.

Functional properties of a disease mutation for migraine in Kv2.1/6.4 channels.

Voltage-gated potassium (Kv) channels are integral to cellular excitability, impacting the resting membrane potential, repolarization, and shaping action potentials in neurons and cardiac myocytes. Structurally, Kv channels are homo or heterotetramers comprising four α-subunits, each with six transmembrane segments (S1-S6). Silent Kv (KvS), includes Kv5.1, Kv6.1-6.4, Kv8.1-8.2, and Kv9.1-9.3, they do not form functional channels on their own but modulate the properties of heteromeric channels. Recent studies have identified the Kv6.4 subunit as a significant modulator within heteromeric channels, such as Kv2.16.4. The Kv2.16.4 heteromer exhibits altered biophysical properties, including a shift in voltage-dependent inactivation and a complex activation. Current genetic studies in migraine patients have revealed a single missense mutation in the Kv6.4 gene. The single missense mutation, L360P is in the highly conserved S4-S5 linker region. This study aims to demonstrate the biophysical effects of the L360P mutation in Kv2.1 6.4 channels with a fixed 2:2 stoichiometry, using monomeric (Kv2.1/6.4) and tandem dimer (Kv2.1_6.4) configurations. Our findings suggest that the L360P mutation significantly impacts the function and regulation of Kv2.1/6.4 channels, providing insights into the molecular mechanisms underlying channel dysfunction in migraine pathology.

Solar-Powered Direct Air Capture: Techno-Economic and Environmental Assessment.

Direct air capture (DAC) of CO2 has gained attention as a sustainable carbon source. One of the most promising technologies currently available is liquid solvent DAC (L-DAC), but the significant fraction of fossil CO2 in the output stream hinders its utilization in carbon-neutral fuels and chemicals. Fossil CO2 is generated and captured during the combustion of fuels to calcine carbonates, which is difficult to decarbonize due to the high temperatures required. Solar thermal energy can provide green high-temperature heat, but it flourishes in arid regions where environmental conditions are typically unfavorable for L-DAC. This study proposes a solar-powered L-DAC approach and develops a model to assess the influence of the location and plant capacity on capture costs. The performed life cycle assessment enables the comparison of technologies based on net CO2 removal, demonstrating that solar-powered L-DAC is not only more environmentally friendly but also more cost-effective than conventional L-DAC.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels.

Cyclic nucleotide-gated (CNG) ion channels of olfactory sensory neurons contain three types of homologue subunits, two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit. Each subunit carries an intracellular cyclic nucleotide binding domain (CNBD) whose occupation by up to four cyclic nucleotides evokes channel activation. Thereby, the subunits interact in a cooperative fashion. Here we studied 16 concatamers with systematically disabled, but still functional, binding sites and quantified channel activation by systems of intimately coupled state models transferred to 4D hypercubes, thereby exploiting a weak voltage dependence of the channels. We provide the complete landscape of free energies for the complex activation process of heterotetrameric channels, including 32 binding steps, in both the closed and open channel, as well as 16 closed-open isomerizations. The binding steps are specific for the subunits and show pronounced positive cooperativity for the binding of the second and the third ligand. The energetics of the closed-open isomerizations were disassembled to elementary subunit promotion energies for channel opening, [Formula: see text], adding to the free energy of the closed-open isomerization of the empty channel, E0. The [Formula: see text] values are specific for the four subunits and presumably invariant for the specific patterns of liganding. In conclusion, subunit cooperativity is confined to the CNBD whereas the subunit promotion energies for channel opening are independent.

Impact of the Sr content on the redox thermodynamics and kinetics of Ca1-xSrxMnO3-δ for tailored properties.

CaMnO3-δ-based perovskites find application in a variety of thermochemical cycles, e.g. oxygen partial pressure adjustment, chemical looping processes, and thermochemical energy storage. The applicability of these materials is governed by their thermodynamic and kinetic properties. Therefore, tunability of these properties is desirable to adapt the material to the required conditions. In this study, the effect of Sr content in Ca1-xSrxMnO3-δ on thermodynamics and kinetics is investigated by thermogravimetric analysis. The thermodynamics are measured in the temperature range of 873 K to 1473 K with oxygen partial pressures of 1 × 10-4 bar to 0.8 bar. The oxidation kinetics were characterized in the temperature range from 473 K to 673 K with oxygen partial pressures of 0.01 bar to 1 bar. The reduction kinetics were very rapid in the temperature range of 873 K to 1023 K, with the measured rates limited by the constraints of the measurement device. The results show that with increasing Sr content the structural changes of the material decrease the reduction enthalpy and the oxidation activation energy. This not only leads to a tunability of material properties, but can also be used to predict changes of these properties when only the structural changes are known.

Technological Pathways to Produce Compressed and Highly Pure Hydrogen from Solar Power.

Hydrogen (H2 ) produced from renewables will have a growing impact on the global energy dynamics towards sustainable and carbon-neutral standards. The share of green H2 is still too low to meet the net-zero target, while the demand for high-quality hydrogen continues to rise. These factors amplify the need for economically viable H2 generation technologies. The present article aims at evaluating the existing technologies for high-quality H2 production based on solar energy. Technologies such as water electrolysis, photoelectrochemical and solar thermochemical water splitting, liquid metal reactors and plasma conversion utilize solar power directly or indirectly (as carbon-neutral electrons) and are reviewed from the perspective of their current development level, technical limitations and future potential.

Enlightening activation gating in P2X receptors.

P2X receptors are trimeric nonselective cation channels gated by ATP. They assemble from seven distinct subunit isoforms as either homo- or heteromeric complexes and contain three extracellularly located binding sites for ATP. P2X receptors are expressed in nearly all tissues and are there involved in physiological processes like synaptic transmission, pain, and inflammation. Thus, they are a challenging pharmacological target. The determination of crystal and cryo-EM structures of several isoforms in the last decade in closed, open, and desensitized states has provided a firm basis for interpreting the huge amount of functional and biochemical data. Electrophysiological characterization in conjugation with optical approaches has generated significant insights into structure-function relationships of P2X receptors. This review focuses on novel optical and related approaches to better understand the conformational changes underlying the activation of these receptors.

Controlling thermal expansion and phase transitions in Ca1-xSrxMnO3-δ by Sr-content.

Perovskite oxides of the general formula ABO3-δ, with A and B being metal cations, present themselves in various crystal structures that originate from a distorted ideal cubic perovskite. Understanding how composition, temperature, atmosphere and reduction extent of these non-stoichiometric redox materials induce structural changes on an atomic, as well as macroscopic, level is crucial to transfer newly developed materials to industrial scale applications in the redox-based energy conversion sector. Herein, Ca1-xSrxMnO3-δ (x ∈ [0,0.2]) and its micro- and macroscopic structural changes at elevated temperatures and varying oxygen partial pressure are analyzed by means of in situ high temperature XRD, DSC and dilatometry. Results are expanded by room temperature XRD of compositions with higher Sr-content up to x = 0.4. By adjusting the Sr-content, the formed crystal structure can be governed and thermal expansion can be impacted beneficially in the context of future applications utilizing monolithic structures. Phase transitions from orthorhombic to cubic were found to shift from 900 °C to 830 °C under air and to even lower temperatures under 1% O2 atmosphere. Small amounts of Sr-content (5-10%) stabilize the macroscopic structural integrity by improving the reversibility of the cyclic thermal expansion and contraction in a 1% O2 atmosphere. However, at Sr-contents of 20% an increased irreversible residual expansion within each thermal cycle becomes apparent and shows that such improvements do not follow a linear dependency with Sr-content, but most benefits in this context can be found at Sr-contents below 20%. The results demonstrate the sensitivity of such materials micro- and macroscopic characteristics to composition. In the context of utilization of monolithic structures, fabricated entirely from Ca1-xSrxMnO3-δ, in thermochemical or thermoelectric applications, the results have considerable significance as minor A-site Sr-substitution can substantially improve macroscopic stability of monolithic structures over multiple thermal cycles. Besides the often solely regarded thermodynamic characteristic, this work demonstrates the importance to consider the impact of composition on structural behavior in materials design processes including perovskites for thermochemical applications.

Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels.

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker (CL) disk. Confocal patch-clamp fluorometry revealed pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL-CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity-theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP-binding sites or toward the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP-binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

Dissecting activation steps in P2X7 receptors.

P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.

Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative.

Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.

Subtype-Specific Ligand Binding and Activation Gating in Homomeric and Heteromeric P2X Receptors.

P2X receptors are ATP-activated, non-specific cation channels involved in sensory signalling, inflammation, and certain forms of pain. Investigations of agonist binding and activation are essential for comprehending the fundamental mechanisms of receptor function. This encompasses the ligand recognition by the receptor, conformational changes following binding, and subsequent cellular signalling. The ATP-induced activation of P2X receptors is further influenced by the concentration of Mg2+ that forms a complex with ATP. To explore these intricate mechanisms, two new fluorescently labelled ATP derivatives have become commercially available: 2-[DY-547P1]-AHT-ATP (fATP) and 2-[DY-547P1]-AHT-α,ßMe-ATP (α,ßMe-fATP). We demonstrate a subtype-specific pattern of ligand potency and efficacy on human P2X2, P2X3, and P2X2/3 receptors with distinct relations between binding and gaiting. Given the high in vivo concentrations of Mg2+, the complex formed by Mg2+ and ATP emerges as an adequate ligand for P2X receptors. Utilising fluorescent ligands, we observed a Mg2+-dependent reduction in P2X2 receptor activation, while binding remained surprisingly robust. In contrast, P2X3 receptors initially exhibited decreased activation at high Mg2+ concentrations, concomitant with increased binding, while the P2X2/3 heteromer showed a hybrid effect. Hence, our new fluorescent ATP derivatives are powerful tools for further unravelling the mechanism underlying ligand binding and activation gating in P2X receptors.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry.

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.

Random mutagenesis identifies factors involved in formate-dependent growth of the methanogenic archaeon Methanococcus maripaludis.

Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO2 to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO2 and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects.

Thermochemical process and compact apparatus for concentrating oxygen in extraterrestrial atmospheres: a feasibility study.

The Martian atmosphere contains 0.16% oxygen, which is an example of an in-situ resource that can be used as precursor or oxidant for propellants, for life support systems and potentially for scientific experiments. Thus, the present work is related to the invention of a process to concentrate oxygen in the oxygen-deficient extraterrestrial atmosphere by means of a thermochemical process and the determination of a suitable best-case apparatus design to carry out the process. The perovskite oxygen pumping (POP) system uses the underlying chemical process, which is based on the temperature-dependent chemical potential of oxygen on multivalent metal oxide, to release and absorb oxygen in response to temperature swings. The primary goal of this work is therefore to identify suitable materials for the oxygen pumping system and to optimize the oxidation-reduction temperature and time, required to operate the system, to produce 2.25 kg of oxygen per hour under the Martian most-extreme environmental conditions and based on the thermochemical process concept. Radioactive materials such as 244Cm, 238Pu and 90Sr are analyzed as a heating source for the operation of the POP system, and critical aspects of the technology as well as weaknesses and uncertainties related to the operational concept are identified.

Kinetic fingerprinting of metabotropic glutamate receptors.

Dimeric metabotropic glutamate receptors (mGluRs) are abundantly expressed in neurons. In mammals, eight subunit isoforms, mGluR1-8, have been identified, forming the groups I, II, and III. We investigated receptor dimerization and kinetics of these mGluR isoforms in excised membrane patches by FRET and confocal patch-clamp fluorometry. We show that 5 out of 8 homodimeric receptors develop characteristic glutamate-induced on- and off-kinetics, as do 11 out of 28 heterodimers. Glutamate-responsive heterodimers were identified within each group, between groups I and II as well as between groups II and III, but not between groups I and III. The glutamate-responsive heterodimers showed heterogeneous activation and deactivation kinetics. Interestingly, mGluR7, not generating a kinetic response in homodimers, showed fast on-kinetics in mGluR2/7 and mGluR3/7 while off-kinetics retained the speed of mGluR2 or mGluR3 respectively. In conclusion, glutamate-induced conformational changes in heterodimers appear within each group and between groups if one group II subunit is present.

A strategy for determining the equilibrium constants for heteromeric ion channels in a complex model.

Ligand-gated ion channels are oligomers containing several binding sites for the ligands. However, the signal transmission from the ligand binding site to the pore has not yet been fully elucidated for any of these channels. In heteromeric channels, the situation is even more complex than in homomeric channels. Using published data for concatamers of heteromeric cyclic nucleotide-gated channels, we show that, on theoretical grounds, multiple functional parameters of the individual subunits can be determined with high precision. The main components of our strategy are (1) the generation of a defined subunit composition by concatenating multiple subunits, (2) the construction of 16 concatameric channels, which differ in systematically permutated binding sites, (3) the determination of respectively differing concentration-activation relationships, and (4) a complex global fit analysis with corresponding intimately coupled Markovian state models. The amount of constraints in this approach is exceedingly high. Furthermore, we propose a stochastic fit analysis with a scaled unitary start vector of identical elements to avoid any bias arising from a specific start vector. Our approach enabled us to determine 23 free parameters, including 4 equilibrium constants for the closed-open isomerizations, 4 disabling factors for the mutations of the different subunits, and 15 virtual equilibrium-association constants in the context of a 4-D hypercube. From the virtual equilibrium-association constants, we could determine 32 equilibrium-association constants of the subunits at different degrees of ligand binding. Our strategy can be generalized and is therefore adaptable to other ion channels.

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels.

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.