Julian Alexander Schreiber

Lysogenic integration of viral genes into host genomes can generate evolutionary pressure that remodels viral protein function, yet the molecular principles underlying such host-driven modulation remain poorly understood. The potassium channel Kesv from Ectocarpus siliculosus virus (Kesv 1) and its host-derived orthologue (Kesv 2) differ by only seven residues but exhibit divergent functional expression. Here, we show that the brown algal host has accumulated pore-adjacent mutations that attenuate channel function by reducing surface expression rather than altering pore architecture. Using electrophysiology, GFP-tagged expression, mutagenesis, and stability simulations, we identify a pair of host-acquired substitutions (Q61H and T66A) that synergistically abolish functional expression despite preserving the canonical selectivity filter, tetramer stability, and overall structural fold. Pharmacological profiling further uncovers noncanonical activation by Sotalol and Linopirdine, establishing Kesv channels as minimalist systems for dissecting core ion channel modulation. Our findings reveal a previously unrecognized evolutionary strategy by which hosts suppress viral ion channel function through subtle, structure-preserving mutations.

K _2P 18.1 (TRESK) displays one of the most unusual two pore domain potassium channels, that is associated with different neurological and immune‐related diseases emphasizing the therapeutic potential of K _2P 18.1 activators. Previous studies identified cloxyquin and nitroxoline as lead compounds that do not alter the function of other K _2P channels. Although their 8‐OH group is crucial for channel activation, 8‐hydroxyquinolines are known for various effects including metal chelation and antibacterial properties increasing the risk of side effects. Therefore, we synthesized 14 quinoline derivatives to search for other tolerated substitutions in 8‐position. Activity determination using two‐electrode voltage clamp (TEVC) revealed that four derivatives achieved moderate inhibitory or stimulatory activity. Among these, benzaldimine 3d represents a new extended scaffold that unexpectedly increases K _2P 18.1 channel activity by about 60% at 100 µM. Docking studies suggest that despite the enlarged structure, 3d is still able to achieve in silico interactions with important amino acids from the cloxyquin binding site. However, its usability in vitro is limited by hydrolysis leading to a compound half‐life of 39 min in aqueous solutions. Nevertheless, the identification of a possible scaffold extension for K _2P 18.1 channel activators opens a new opportunity for future compound diversification.

In the 1950s, Hodgkin and Huxley predicted the existence of ion channels that produce a continuous potassium (K⁺) background current, maintaining the resting membrane potential. In the mid-1990s, these leak channels were identified and named two-pore domain potassium channels (K_2P) based on their unique monomer structure. In a very short period, 15 different genes encoding for K_2P channel subunits were identified within the human genome that form functional homo as well as heterodimeric K⁺ channels. Based on their functional properties, homodimeric K_2P channels were subdivided into the six different subfamilies TWIK, TREK, TASK, TALK, THIK, and TRESK. Intensive research uncovered the critical contribution of K_2P channels to basal physiological processes in all kinds of different tissues. Since 2012, functional studies have been aligned with structural examinations via crystallography and cryo-electron microscopy. The combination of functional and structural research generates a deeper understanding for physiological as well as pathophysiological processes supporting the development of specific ion channel modulators. Hereafter, we will recapitulate the current knowledge from a structural and functional perspective, focusing on the different domains of K_2P ion channels as well as their contribution to certain physiological processes.

Background/Aims: Over the years, the number of patients with neurodegenerative diseases is constantly rising illustrating the need for new neuroprotective drugs. A promising treatment approach is the reduction of excitotoxicity induced by rising (S)-glutamate levels and subsequent NMDA receptor overactivation. To facilitate the search for new NMDA receptor inhibitors neuronal cell models are needed. In this study, we evaluated the suitability of human SK-N-SH cells to serve as a cell model for neurodegeneration induced by NMDA receptor overstimulation. Methods: The cytoprotective effect of the unselective NMDA receptor blocker ketamine as well as the GluN2B-selective inhibitor WMS14-10 was evaluated utilizing different cell viability assays, such as endpoint (LDH, CCK-8, DAPI/FACS) and time dependent methods (bioimpedance). Results: Non-differentiated as well as differentiated SK-N-SH cells express GluN1 and GluN2B subunits. Furthermore, 50 mM (S)-glutamate led to an instantaneous decrease in cell survival. Only application of unselective channel blocker ketamine could protect differentiated cells against this effect, while the selective inhibitor WMS14-10 did not significantly increase cell survival. Conclusion: SK-N-SH cells show an increased sensitivity to (S)-glutamate mediated cytotoxicity with higher differentiation level, that is only partially induced by NMDA receptor overstimulation. Furthermore, we showed that only unselective NMDA receptor inhibition can partially reverse (S)-glutamate-induced toxicity.

The TWIK-related spinal cord K⁺ channel (TRESK, K_2P18.1) is a K_2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K⁺ / Rb⁺). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

The TWIK-related spinal cord K⁺ channel (TRESK) is a K_2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular mechanism causing the agonism can foster the future drug development. Here, we identified the cloxyquin binding site at the M2/M4 interface by mutational scan and analyzed the molecular mechanism of action by protein modelling as well as in silico and in vitro electrophysiology using different permeating ion species (K⁺ / Rb⁺). In combination with kinetic analyses of channel inactivation, we show that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.

During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1 and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication.

The TWIK-related spinal cord K+ channel (TRESK) is part of the two-pore domain K+ channel family (K2P), which are also called leak potassium channels. As indicated by the channel family name, TRESK conducts K+ ions along the concentration gradient in a nearly voltage-independent manner leading to lowered membrane potentials. Although functional and pharmacological similarities exist, TRESK shows low sequence identity with other K2P channels. Moreover, the channel possesses several unique features such as its sensitivity to intracellular Ca2+ ions, that are not found in other K2P channels. High expression rates are found in immune-associated and neuronal cells, especially in sensory neurons of the dorsal root and trigeminal ganglia. As a consequence of the induced hyperpolarization, TRESK influences neuronal firing, the release of inflammatory mediators and the proliferation of distinct immune cells. Consequently, this channel might be a suitable target for pharmacological intervention in migraine, epilepsy, neuropathic pain or distinct immune diseases. In this review, we summarize the biochemical and biophysical properties of TRESK channels as well as their sensitivity to different known compounds. Furthermore, we give a structured overview about the physiological and pathophysiological impact of TRESK, that render the channel as an interesting target for specific drug development.

GluN2B-NMDA receptors play a key role in several neurological and neurodegenerative disorders. In order to develop novel negative allosteric GluN2B-NMDA receptor modulators, the concept of conformational restriction was pursued, i.e. the flexible aminoethanol substructure of ifenprodil was embedded into a more rigid tetrahydro-3-benzazepine system. The resulting tetrahydro-3-benzazepine-1,7-diol (±)-2 (WMS-1410) showed promising receptor affinity in receptor binding studies (K _i = 84 nM) as well as pharmacological activity in two-electrode-voltage-clamp experiments (IC ₅₀ = 116 nM) and in cytoprotective assays (IC ₅₀ = 18.5 nM). The interactions of (R)-2 with the ifenprodil binding site of GluN2B-NMDA receptors were analyzed on the molecular level and the “foot-in-the-door” mechanism was developed. Due to promising pharmacokinetic parameters (logD_7.4 = 1.68, plasma protein binding of 76–77%, sufficient metabolic stability) F-substituted analogs were prepared and evaluated as tracers for positron emission tomography (PET). Both fluorine-18-labeled PET tracers [¹⁸F]11 and [¹⁸F]15 showed high brain uptake, specific accumulation in regions known for high GluN2B-NMDA receptor expression, but no interactions with σ ₁ receptors. Radiometabolites were not observed in the brain. Both PET tracers might be suitable for application in humans.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (I_h) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of I_h (potential of half-maximal activation (V_0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, I_h was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that I_h parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V_0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.