Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells.

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future.

Hippocampal neurons maintain a large PtdIns(4)P pool that results in faster PtdIns(4,5)P2 synthesis.

PtdIns(4,5)P2 is a signaling lipid central to the regulation of multiple cellular functions. It remains unknown how PtdIns(4,5)P2 fulfills various functions in different cell types, such as regulating neuronal excitability, synaptic release, and astrocytic function. Here, we compared the dynamics of PtdIns(4,5)P2 synthesis in hippocampal neurons and astrocytes with the kidney-derived tsA201 cell line. The experimental approach was to (1) measure the abundance and rate of PtdIns(4,5)P2 synthesis and precursors using specific biosensors, (2) measure the levels of PtdIns(4,5)P2 and its precursors using mass spectrometry, and (3) use a mathematical model to compare the metabolism of PtdIns(4,5)P2 in cell types with different proportions of phosphoinositides. The rate of PtdIns(4,5)P2 resynthesis in hippocampal neurons after depletion by cholinergic or glutamatergic stimulation was three times faster than for tsA201 cells. In tsA201 cells, resynthesis of PtdIns(4,5)P2 was dependent on the enzyme PI4K. In contrast, in hippocampal neurons, the resynthesis rate of PtdIns(4,5)P2 was insensitive to the inhibition of PI4K, indicating that it does not require de novo synthesis of the precursor PtdIns(4)P. Measurement of phosphoinositide abundance indicated a larger pool of PtdIns(4)P, suggesting that hippocampal neurons maintain sufficient precursor to restore PtdIns(4,5)P2 levels. Quantitative modeling indicates that the measured differences in PtdIns(4)P pool size and higher activity of PI4K can account for the experimental findings and indicates that high PI4K activity prevents depletion of PtdIns(4)P. We further show that the resynthesis of PtdIns(4,5)P2 is faster in neurons than astrocytes, providing context to the relevance of cell type-specific mechanisms to sustain PtdIns(4,5)P2 levels.

Translational investigation of electrophysiology in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) patients are at increased risk of ventricular arrhythmias and sudden cardiac death, which can occur even in the absence of structural changes of the heart. HCM mouse models suggest mutations in myofilament components to affect Ca2+ homeostasis and thereby favor arrhythmia development. Additionally, some of them show indications of pro-arrhythmic changes in cardiac electrophysiology. In this study, we explored arrhythmia mechanisms in mice carrying a HCM mutation in Mybpc3 (Mybpc3-KI) and tested the translatability of our findings in human engineered heart tissues (EHTs) derived from CRISPR/Cas9-generated homozygous MYBPC3 mutant (MYBPC3hom) in induced pluripotent stem cells (iPSC) and to left ventricular septum samples obtained from HCM patients. We observed higher arrhythmia susceptibility in contractility measurements of field-stimulated intact cardiomyocytes and ventricular muscle strips as well as in electromyogram recordings of Langendorff-perfused hearts from adult Mybpc3-KI mice than in wild-type (WT) controls. The latter only occurred in homozygous (Hom-KI) but not in heterozygous (Het-KI) mouse hearts. Both Het- and Hom-KI are known to display pro-arrhythmic increased Ca2+ myofilament sensitivity as a direct consequence of the mutation. In the electrophysiological characterization of the model, we observed smaller repolarizing K+ currents in single cell patch clamp, longer ventricular action potentials in sharp microelectrode recordings and longer ventricular refractory periods in Langendorff-perfused hearts in Hom-KI, but not Het-KI. Interestingly, reduced K+ channel subunit transcript levels and prolonged action potentials were already detectable in newborn, pre-hypertrophic Hom-KI mice. Human iPSC-derived MYBPC3hom EHTs, which genetically mimicked the Hom-KI mice, did exhibit lower mutant mRNA and protein levels, lower force, beating frequency and relaxation time, but no significant alteration of the force-Ca2+ relation in skinned EHTs. Furthermore, MYBPC3hom EHTs did show higher spontaneous arrhythmic behavior, whereas action potentials measured by sharp microelectrode did not differ to isogenic controls. Action potentials measured in septal myectomy samples did not differ between patients with HCM and patients with aortic stenosis, except for the only sample with a MYBPC3 mutation. The data demonstrate that increased myofilament Ca2+ sensitivity is not sufficient to induce arrhythmias in the Mybpc3-KI mouse model and suggest that reduced K+ currents can be a pro-arrhythmic trigger in Hom-KI mice, probably already in early disease stages. However, neither data from EHTs nor from left ventricular samples indicate relevant reduction of K+ currents in human HCM. Therefore, our study highlights the species difference between mouse and human and emphasizes the importance of research in human samples and human-like models.

Control of Neuronal Excitability by Cell Surface Receptor Density and Phosphoinositide Metabolism.

Phosphoinositides are members of a family of minor phospholipids that make up about 1% of all lipids in most cell types. Despite their low abundance they have been found to be essential regulators of neuronal activities such as action potential firing, release and re-uptake of neurotransmitters, and interaction of cytoskeletal proteins with the plasma membrane. Activation of several different neurotransmitter receptors can deplete phosphoinositide levels by more than 90% in seconds, thereby profoundly altering neuronal behavior; however, despite the physiological importance of this mechanism we still lack a profound quantitative understanding of the connection between phosphoinositide metabolism and neuronal activity. Here, we present a model that describes phosphoinositide metabolism and phosphoinositide-dependent action potential firing in sympathetic neurons. The model allows for a simulation of activation of muscarinic acetylcholine receptors and its effects on phosphoinositide levels and their regulation of action potential firing in these neurons. In this paper, we describe the characteristics of the model, its calibration to experimental data, and use the model to analyze how alterations of surface density of muscarinic acetylcholine receptors or altered activity levels of a key enzyme of phosphoinositide metabolism influence action potential firing of sympathetic neurons. In conclusion, the model provides a comprehensive framework describing the connection between muscarinic acetylcholine signaling, phosphoinositide metabolism, and action potential firing in sympathetic neurons which can be used to study the role of these signaling systems in health and disease.

Cytosine-Based TET Enzyme Inhibitors.

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-µM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.

Reinterpretation of the substrate specificity of the voltage-sensing phosphatase during dimerization.

Voltage-sensing phosphatases (VSPs) cleave both 3- and 5-phosphates from inositol phospholipids in response to membrane depolarization. When low concentrations of Ciona intestinalis VSP are expressed in Xenopus laevis oocytes, the 5-phosphatase reaction can be observed during large membrane depolarizations. When higher concentrations are expressed, the 5-phosphatase activity is observed with smaller depolarizations, and the 3-phosphatase activity is revealed with strong depolarization. Here we ask whether this apparent induction of 3-phosphatase activity is attributable to the dimerization that has been reported when VSP is expressed at higher concentrations. Using a simple kinetic model, we show that these enzymatic phenomena can be understood as an emergent property of a voltage-dependent enzyme with invariant substrate selectivity operating in the context of endogenous lipid-metabolizing enzymes present in oocytes. Thus, a switch of substrate specificity with dimerization need not be invoked to explain the appearance of 3-phosphatase activity at high VSP concentrations.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Fatty-acyl chain profiles of cellular phosphoinositides.

Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2 pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in <20s of both PIP2 and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.

Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence.

Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P3 is 55-fold slower than 5-phosphatase activity against PI(4,5)P2; thus, PI(4,5)P2 generated more slowly from dephosphorylating PI(3,4,5)P3 might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation, emphasizing that VSPs can cleave the 3-phosphate of PI(3,4,5)P3.

Osmoregulatory inositol transporter SMIT1 modulates electrical activity by adjusting PI(4,5)P2 levels.

Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na(+)/myo-inositol cotransporter (SMIT1) and myo-inositol supplementation enlarged intracellular PI(4,5)P2 pools, modulated several PI(4,5)P2-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myo-inositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP2 were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several "physiological" manipulations that alter the PI(4,5)P2 setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells.

Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides.

Endoplasmic reticulum-plasma membrane (ER-PM) contact sites play an integral role in cellular processes such as excitation-contraction coupling and store-operated calcium entry (SOCE). Another ER-PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER-PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2 Activation of G protein-coupled receptors that deplete PM PI(4,5)P2disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1's access to the PM and permitting PM PI(4)P and PI(4,5)P2to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER-PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.

GABAergic signaling in the rat pineal gland.

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ-aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABAA receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABAB 1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABAA receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage-gated Ca(2+) channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48-72-h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine-induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.

Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons.

In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca(2+) upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons.

Phosphoinositides regulate ion channels.

Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. This article is part of a Special Issue entitled Phosphoinositides.

Nerve growth factor sensitizes adult sympathetic neurons to the proinflammatory peptide bradykinin.

Levels of nerve growth factor (NGF) are elevated in inflamed tissues. In sensory neurons, increases in NGF augment neuronal sensitivity (sensitization) to noxious stimuli. Here, we hypothesized that NGF also sensitizes sympathetic neurons to proinflammatory stimuli. We cultured superior cervical ganglion (SCG) neurons from adult male Sprague Dawley rats with or without added NGF and compared their responsiveness to bradykinin, a proinflammatory peptide. The NGF-cultured neurons exhibited significant depolarization, bursts of action potentials, and Ca(2+) elevations after bradykinin application, whereas neurons cultured without NGF showed only slight changes in membrane potential and cytoplasmic Ca(2+) levels. The NGF effect, which requires trkA receptors, takes hours to develop and days to reverse. We addressed the ionic mechanisms underlying this sensitization. NGF did not alter bradykinin-induced M-current inhibition or phosphatidylinositol 4,5-bisphosphate hydrolysis. Maxi-K channel-mediated current evoked by depolarizations was reduced by 50% by culturing neurons in NGF. Application of iberiotoxin or paxilline, blockers of Maxi-K channels, mimicked NGF treatment and sensitized neurons to bradykinin application. A calcium channel blocker also mimicked NGF treatment. We found that NGF reduces Maxi-K channel opening by decreasing the activity of nifedipine-sensitive calcium channels. In conclusion, culture in NGF reduces the activity of L-type calcium channels, and secondarily, the calcium-sensitive activity of Maxi-K channels, rendering sympathetic neurons electrically hyper-responsive to bradykinin.

TRPM4 channels in the cardiovascular system.

The non-selective Transient Receptor Potential Melastatin 4 (TRPM4) cation channel is abundantly expressed in cardiac cells, being involved in several aspects of cardiac rhythmicity, including cardiac conduction, pace making and action-potential repolarization. Dominantly inherited mutations in the TRPM4 gene are associated with the cardiac bundle-branch disorder progressive familial heart block type I (PFHBI) and isolated cardiac conduction disease (ICCD) giving rise to atrio-ventricular conduction block (AVB), right bundle branch block, bradycardia, and the Brugada syndrome. The mutant phenotypes closely resemble those associated with mutations in the SCN5A gene, encoding the voltage-gated Na(+) channel NaV1.5. These observations and the unexpected partnership with sulfonylurea-receptors (SURs) makes the TRPM4 channel a promising novel target for treatment of cardiac disorders.

Dynamic metabolic control of an ion channel.

G-protein-coupled receptors mediate responses to external stimuli in various cell types. We are interested in the modulation of KCNQ2/3 potassium channels by the Gq-coupled M1 muscarinic (acetylcholine) receptor (M1R). Here, we describe development of a mathematical model that incorporates all known steps along the M1R signaling cascade and accurately reproduces the macroscopic behavior we observe when KCNQ2/3 currents are inhibited following M1R activation. Gq protein-coupled receptors of the plasma membrane activate phospholipase C (PLC) which cleaves the minor plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) into the second messengers diacylgycerol and inositol 1,4,5-trisphosphate, leading to calcium release, protein kinase C (PKC) activation, and PI(4,5)P2 depletion. Combining optical and electrical techniques with knowledge of relative abundance of each signaling component has allowed us to develop a kinetic model and determine that (i) M1R activation and M1R/Gß interaction are fast; (ii) Gαq/Gß separation and Gαq/PLC interaction have intermediate time constants; (iii) the amount of activated PLC limits the rate of KCNQ2/3 suppression; (iv) weak PLC activation can elicit robust calcium signals without net PI(4,5)P2 depletion or KCNQ2/3 channel inhibition; and (v) depletion of PI(4,5)P2, and not calcium/CaM or PKC-mediated phosphorylation, closes KCNQ2/3 potassium channels, thereby increasing neuronal excitability.

The MoSGrid Science Gateway - A Complete Solution for Molecular Simulations.

The MoSGrid portal offers an approach to carry out high-quality molecular simulations on distributed compute infrastructures to scientists with all kinds of background and experience levels. A user-friendly Web interface guarantees the ease-of-use of modern chemical simulation applications well established in the field. The usage of well-defined workflows annotated with metadata largely improves the reproducibility of simulations in the sense of good lab practice. The MoSGrid science gateway supports applications in the domains quantum chemistry (QC), molecular dynamics (MD), and docking. This paper presents the open-source MoSGrid architecture as well as lessons learned from its design.

The phosphoinositide sensitivity of the K(v) channel family.

Recently, we screened several KV channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2). The channels were expressed in tsA-201 cells and the PI(4,5)P 2 was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only KV 7, but none of the other tested KV channels, to be strongly dependent on PI(4,5)P 2. We now have extended our study to KV 1.2 channels, a KV channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P 2. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P 2 and a small left shift in the activation curve of KV 1.2 channels. We also found a small reduction of KV 11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P 2-dependence of KV 1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P 2 sensitivity of some ion channels and insensitivity of others.

TRPM4 cation channel mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis.

In multiple sclerosis, an inflammatory disease of the central nervous system (CNS), axonal and neuronal loss are major causes for irreversible neurological disability. However, which molecules contribute to axonal and neuronal injury under inflammatory conditions remains largely unknown. Here we show that the transient receptor potential melastatin 4 (TRPM4) cation channel is crucial in this process. TRPM4 is expressed in mouse and human neuronal somata, but it is also expressed in axons in inflammatory CNS lesions in experimental autoimmune encephalomyelitis (EAE) in mice and in human multiple sclerosis tissue. Deficiency or pharmacological inhibition of TRPM4 using the antidiabetic drug glibenclamide resulted in reduced axonal and neuronal degeneration and attenuated clinical disease scores in EAE, but this occurred without altering EAE-relevant immune function. Furthermore, Trpm4(-/-) mouse neurons were protected against inflammatory effector mechanisms such as excitotoxic stress and energy deficiency in vitro. Electrophysiological recordings revealed TRPM4-dependent neuronal ion influx and oncotic cell swelling upon excitotoxic stimulation. Therefore, interference with TRPM4 could translate into a new neuroprotective treatment strategy.