Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum.

When the K+ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.

SAFit2 reduces neuroinflammation and ameliorates nerve injury-induced neuropathic pain.

BACKGROUND: Neuropathic pain is experienced worldwide by patients suffering from nerve injuries, infectious or metabolic diseases or chemotherapy. However, the treatment options are still limited because of low efficacy and sometimes severe side effects. Recently, the deficiency of FKBP51 was shown to relieve chronic pain, revealing FKBP51 as a potential therapeutic target. However, a specific and potent FKBP51 inhibitor was not available until recently which hampered targeting of FKBP51. METHODS: In this study, we used the well-established and robust spared nerve injury model to analyze the effect of SAFit2 on nerve injury-induced neuropathic pain and to elucidate its pharmacodynamics profile. Therefore, the mice were treated with 10 mg/kg SAFit2 after surgery, the mice behavior was assessed over 21 days and biochemical analysis were performed after 14 and 21 days. Furthermore, the impact of SAFit2 on sensory neurons and macrophages was investigated in vitro. RESULTS: Here, we show that the FKBP51 inhibitor SAFit2 ameliorates nerve injury-induced neuropathic pain in vivo by reducing neuroinflammation. SAFit2 reduces the infiltration of immune cells into neuronal tissue and counteracts the increased NF-κB pathway activation which leads to reduced cytokine and chemokine levels in the DRGs and spinal cord. In addition, SAFit2 desensitizes the pain-relevant TRPV1 channel and subsequently reduces the release of pro-inflammatory neuropeptides from sensory neurons. CONCLUSIONS: SAFit2 ameliorates neuroinflammation and counteracts enhanced neuronal activity after nerve injury leading to an amelioration of nerve injury-induced neuropathic pain. Based on these findings, SAFit2 constitutes as a novel and promising drug candidate for the treatment of nerve injury-induced neuropathic pain.

Nonparametric Bayesian inference for meta-stable conformational dynamics.

Analyses of structural dynamics of biomolecules hold great promise to deepen the understanding of and ability to construct complex molecular systems. To this end, both experimental and computational means are available, such as fluorescence quenching experiments or molecular dynamics simulations, respectively. We argue that while seemingly disparate, both fields of study have to deal with the same type of data about the same underlying phenomenon of conformational switching. Two central challenges typically arise in both contexts: (i) the amount of obtained data is large, and (ii) it is often unknown how many distinct molecular states underlie these data. In this study, we build on the established idea of Markov state modeling and propose a generative, Bayesian nonparametric hidden Markov state model that addresses these challenges. Utilizing hierarchical Dirichlet processes, we treat different meta-stable molecule conformations as distinct Markov states, the number of which we then do not have to seta priori. In contrast to existing approaches to both experimental as well as simulation data that are based on the same idea, we leverage a mean-field variational inference approach, enabling scalable inference on large amounts of data. Furthermore, we specify the model also for the important case of angular data, which however proves to be computationally intractable. Addressing this issue, we propose a computationally tractable approximation to the angular model. We demonstrate the method on synthetic ground truth data and apply it to known benchmark problems as well as electrophysiological experimental data from a conformation-switching ion channel to highlight its practical utility.

Cell Volume Regulation in the Epidermis.

In order to cope with external stressors such as changes in humidity and temperature or irritating substances, the epidermis as the outermost skin layer forms a continuously renewing and ideally intact protective barrier. Under certain circumstances, this barrier can be impaired and epidermal cells have to counteract cell swelling or shrinkage induced by osmotic stress via regulatory volume decrease (RVD) or increase (RVI). Here, we will review the current knowledge regarding the molecular machinery underlying RVD and RVI in the epidermis. Furthermore, we will discuss the current understanding how cell volume changes and its regulators are associated with epidermal renewal and barrier formation.

Characterization of an N-terminal Nav1.5 channel variant - a potential risk factor for arrhythmias and sudden death?

BACKGROUND: Alterations in the SCN5A gene encoding the cardiac sodium channel Nav1.5 have been linked to a number of arrhythmia syndromes and diseases including long-QT syndrome (LQTS), Brugada syndrome (BrS) and dilative cardiomyopathy (DCM), which may predispose to fatal arrhythmias and sudden death. We identified the heterozygous variant c.316A > G, p.(Ser106Gly) in a 35-year-old patient with survived cardiac arrest. In the present study, we aimed to investigate the functional impact of the variant to clarify the medical relevance. METHODS: Mutant as well as wild type GFP tagged Nav1.5 channels were expressed in HEK293 cells. We performed functional characterization experiments using patch-clamp technique. RESULTS: Electrophysiological measurements indicated, that the detected missense variant alters Nav1.5 channel functionality leading to a gain-of-function effect. Cells expressing S106G channels show an increase in Nav1.5 current over the entire voltage window. CONCLUSION: The results support the assumption that the detected sequence aberration alters Nav1.5 channel function and may predispose to cardiac arrhythmias and sudden cardiac death.

Fusicoccin Activates KAT1 Channels by Stabilizing Their Interaction with 14-3-3 Proteins.

Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K+ channels. Here, we present crystallographic and functional data showing that the K+ inward rectifier KAT1 (K+Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco (Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K+ uptake in plant cells.

Identification of Intrahelical Bifurcated H-Bonds as a New Type of Gate in K+ Channels.

Gating of ion channels is based on structural transitions between open and closed states. To uncover the chemical basis of individual gates, we performed a comparative experimental and computational analysis between two K+ channels, KcvS and KcvNTS. These small viral encoded K+ channel proteins, with a monomer size of only 82 amino acids, resemble the pore module of all complex K+ channels in terms of structure and function. Even though both proteins share about 90% amino acid sequence identity, they exhibit different open probabilities with ca. 90% in KcvNTS and 40% in KcvS. Single channel analysis, mutational studies and molecular dynamics simulations show that the difference in open probability is caused by one long closed state in KcvS. This state is structurally created in the tetrameric channel by a transient, Ser mediated, intrahelical hydrogen bond. The resulting kink in the inner transmembrane domain swings the aromatic rings from downstream Phes in the cavity of the channel, which blocks ion flux. The frequent occurrence of Ser or Thr based helical kinks in membrane proteins suggests that a similar mechanism could also occur in the gating of other ion channels.

The small neurotoxin apamin blocks not only small conductance Ca2+ activated K+ channels (SK type) but also the voltage dependent Kv1.3 channel.

Apamin is frequently used as a specific blocker of small-conductance Ca2+-activated (SK type) K+ channels. Here we show that the small neurotoxin is not as specific as anticipated. It is also a high-affinity inhibitor with an IC50 of 13 nM of the Kv1.3 channel; it blocks the latter with potency similar to the Kv1.3 blocker PAP-1. Since SK type channels and Kv1.3 channels are frequently coexpressed in different tissues such as cells of the immune system, apamin must be used with caution as a pharmacological tool.

Mutation in pore-helix modulates interplay between filter gate and Ba2+ block in a Kcv channel pore.

The selectivity filter of K+ channels catalyzes a rapid and highly selective transport of K+ while serving as a gate. To understand the control of this filter gate, we use the pore-only K+ channel KcvNTS in which gating is exclusively determined by the activity of the filter gate. It has been previously shown that a mutation at the C-terminus of the pore-helix (S42T) increases K+ permeability and introduces distinct voltage-dependent and K+-sensitive channel closures at depolarizing voltages. Here, we report that the latter are not generated by intrinsic conformational changes of the filter gate but by a voltage-dependent block caused by nanomolar trace contaminations of Ba2+ in the KCl solution. Channel closures can be alleviated by extreme positive voltages and they can be completely abolished by the high-affinity Ba2+ chelator 18C6TA. By contrast, the same channel closures can be augmented by adding Ba2+ at submicromolar concentrations to the cytosolic buffer. These data suggest that a conservative exchange of Ser for Thr in a crucial position of the filter gate increases the affinity of the filter for Ba2+ by >200-fold at positive voltages. While Ba2+ ions apparently remain only for a short time in the filter-binding sites of the WT channel before passing the pore, they remain much longer in the mutant channel. Our findings suggest that the dwell times of permeating and blocking ions in the filter-binding sites are tightly controlled by interactions between the pore-helix and the selectivity filter.

Binding kinetics of quaternary ammonium ions in Kcv potassium channels.

Kcv channels from plant viruses represent the autonomous pore module of potassium channels, devoid of any regulatory domains. These small proteins show very reproducible single-channel behavior in planar lipid bilayers. Thus, they are an optimum system for the study of the biophysics of ion transport and gating. Structural models based on homology modeling have been used successfully, but experimental structural data are currently not available. Here we determine the size of the cytosolic pore entrance by studying the blocker kinetics. Blocker binding and dissociation rate constants ranging from 0.01 to 1000 ms-1 were determined for different quaternary ammonium ions. We found that the cytosolic pore entrance of KcvNTS must be at least 11 Å wide. The results further indicate that the residues controlling a cytosolic gate in one of the Kcv isoforms influence blocker binding/dissociation as well as a second gate even when the cytosolic gate is in the open state. The voltage dependence of the rate constant of blocker release is used to test, which blockers bind to the same binding site.

Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K+-Responsive Fluorescent Protein Sensor.

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focusing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors and now provides a capacity to assess the specificity of a nanopore or ion channel. Finally, a comparison to high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.

Alkali metal cations modulate the geometry of different binding sites in HCN4 selectivity filter for permeation or block.

Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.

Tailoring baker's yeast Saccharomyces cerevisiae for functional testing of channelrhodopsin.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker's yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines.

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4-/- knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4-/- cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

Perturbation of the host cell Ca2+ homeostasis and ER-mitochondria contact sites by the SARS-CoV-2 structural proteins E and M.

Coronavirus disease (COVID-19) is a contagious respiratory disease caused by the SARS-CoV-2 virus. The clinical phenotypes are variable, ranging from spontaneous recovery to serious illness and death. On March 2020, a global COVID-19 pandemic was declared by the World Health Organization (WHO). As of February 2023, almost 670 million cases and 6,8 million deaths have been confirmed worldwide. Coronaviruses, including SARS-CoV-2, contain a single-stranded RNA genome enclosed in a viral capsid consisting of four structural proteins: the nucleocapsid (N) protein, in the ribonucleoprotein core, the spike (S) protein, the envelope (E) protein, and the membrane (M) protein, embedded in the surface envelope. In particular, the E protein is a poorly characterized viroporin with high identity amongst all the ß-coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43) and a low mutation rate. Here, we focused our attention on the study of SARS-CoV-2 E and M proteins, and we found a general perturbation of the host cell calcium (Ca2+) homeostasis and a selective rearrangement of the interorganelle contact sites. In vitro and in vivo biochemical analyses revealed that the binding of specific nanobodies to soluble regions of SARS-CoV-2 E protein reversed the observed phenotypes, suggesting that the E protein might be an important therapeutic candidate not only for vaccine development, but also for the clinical management of COVID designing drug regimens that, so far, are very limited.

Role of Ion Distribution and Energy Barriers for Concerted Motion of Subunits in Selectivity Filter Gating of a K+ Channel.

Most potassium channels have two main gate locations, hosting an inner gate at the cytosolic entrance and a filter gate in the selectivity filter; the function of these gates is in many channels coupled. To obtain exclusive insights into the molecular mechanisms that determine opening and closing of the filter gate, we use a combination of single-channel recordings and gating analysis in the minimal viral channel KcvNTS. This channel has no inner gate, and its fast closing at negative voltages can therefore be entirely assigned to the filter gate. We find that mutations of S42 in the pore helix severely slow down closing of this filter gate, an effect which is not correlated with hydrogen bond formation by the amino acid at this position. Hence, different from KcsA, which contains the critical E71 in the equivalent position forming a salt bridge, the coupling between selectivity filter and surrounding structures for filter gating must in KcvNTS rely on different modes of interaction. Quantitative analysis of concatemers carrying different numbers of S42T mutations reveals that each subunit contributes the same amount of âˆ¼ 0.4 kcal/mol to the energy barrier for filter closure indicating a concerted action of the subunits. Since the mutations have neither an influence on the unitary current nor on the voltage dependency of the gate, the data stress that the high subunit cooperativity is mediated through conformational changes rather than through changes in the ion occupation in the selectivity filter.

Combining in vitro translation with nanodisc technology and functional reconstitution of channels in planar lipid bilayers.

Experimental studies on membrane proteins have been recently enriched by two promising method developments: protocols for cell-free protein synthesis and the use of soluble nanoscale lipid bilayers, so called nanodiscs, as membrane mimics for keeping these proteins in a soluble form. Here, we show how the advantages of these techniques can be combined with the classical planar lipid bilayer method for a functional reconstitution of channel activity. The present data demonstrate that the combination of these methods offers a very rapid and reliable way of recording channel activity in different bilayer systems. This approach has additional advantages in that it strongly lowers the propensity of contamination from the expression system and allows the simultaneous reconstitution of thousands of channel proteins for macroscopic current measurements without compromising bilayer stability.

A Human 3D Cardiomyocyte Risk Model to Study the Cardiotoxic Influence of X-rays and Other Noxae in Adults.

The heart tissue is a potential target of various noxae contributing to the onset of cardiovascular diseases. However, underlying pathophysiological mechanisms are largely unknown. Human stem cell-derived models are promising, but a major concern is cell immaturity when estimating risks for adults. In this study, 3D aggregates of human embryonic stem cell-derived cardiomyocytes were cultivated for 300 days and characterized regarding degree of maturity, structure, and cell composition. Furthermore, effects of ionizing radiation (X-rays, 0.1-2 Gy) on matured aggregates were investigated, representing one of the noxae that are challenging to assess. Video-based functional analyses were correlated to changes in the proteome after irradiation. Cardiomyocytes reached maximum maturity after 100 days in cultivation, judged by α-actinin lengths, and displayed typical multinucleation and branching. At this time, aggregates contained all major cardiac cell types, proven by the patch-clamp technique. Matured and X-ray-irradiated aggregates revealed a subtle increase in beat rates and a more arrhythmic sequence of cellular depolarisation and repolarisation compared to non-irradiated sham controls. The proteome analysis provides first insights into signaling mechanisms contributing to cardiotoxicity. Here, we propose an in vitro model suitable to screen various noxae to target adult cardiotoxicity by preserving all the benefits of a 3D tissue culture.

Asymmetric Interplay Between K+ and Blocker and Atomistic Parameters From Physiological Experiments Quantify K+ Channel Blocker Release.

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels.

Codon Bias Can Determine Sorting of a Potassium Channel Protein.

Due to the redundancy of the genetic code most amino acids are encoded by multiple synonymous codons. It has been proposed that a biased frequency of synonymous codons can affect the function of proteins by modulating distinct steps in transcription, translation and folding. Here, we use two similar prototype K+ channels as model systems to examine whether codon choice has an impact on protein sorting. By monitoring transient expression of GFP-tagged channels in mammalian cells, we find that one of the two channels is sorted in a codon and cell cycle-dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves, together with a cell-state-dependent decoding mechanism, as a secondary code for sorting intracellular membrane proteins.