Fast Activation Kinetics Research Articles

Illumination of Hydroxyl Radical in Kidney Injury and High-Throughput Screening of Natural Protectants Using a Fluorescent/Photoacoustic Probe.

The hydroxyl radical (•OH) is shown to play a crucial role in the occurrence and progression of acute kidney injury (AKI). Therefore, the development of a robust •OH probe holds great promise for the early diagnosis of AKI, high-throughput screening (HTS) of natural protectants, and elucidating the molecular mechanism of intervention in AKI. Herein, the design and synthesis of an activatable fluorescent/photoacoustic (PA) probe (CDIA) for sensitive and selective imaging of •OH in AKI is reported. CDIA has near-infrared fluorescence/PA channels and fast activation kinetics, enabling the detection of the onset of •OH in an AKI model. The positive detection time of 12 h using this probe is superior to the 48-hour detection time for typical clinical assays, such as blood urea nitrogen and serum creatinine detection. Furthermore, a method is established using CDIA for HTS of natural •OH inhibitors from herbal medicines. Puerarin is screened out by activating the Sirt1/Nrf2/Keap1 signaling pathway to protect renal cells in AKI. Overall, this work provides a versatile and dual-mode tool for illuminating the •OH-related pathological process in AKI and screening additional compounds to prevent and treat AKI.

Modulation of delayed rectifying K+ currents by silent Kv subunits.

Voltage-gated potassium channels (VGKCs) play fundamental roles in neurobiology; they are a major hub for potential modulation of cell excitability and health. VGKCs are formed by homotetramerization of K+ channels subunits (Kv1-12). Kv2 channels are the major cause of the delayed rectifying K+ currents (IKdr) that tune neuronal firing frequency. Uniquely, Kv2 subunits can co-assemble with members of four other families of Kv (Kv5-6, 8-9). These other Kvs are named silent since they do not form functional channels themselves as homomeric VGKCs. Heteromeric co-assemblies (hetVGKCs) form biophysically distinct channels that modify IKdr and therefore determine neuronal excitability. More than that, the expression of silent subunits suppresses the overall expression of Kv2 subunits, affecting the abundancy of homomeric Kv2 channels and heteromeric channels containing Kv2 subunits. To date, no established mechanism explains how silent Kv subunits interfere with hetVGKCs gating processes or how the mechanism of suppression works. Here, we used Kv6.4 as archetypal silent subunit expressed together with Kv2.1 in HEK cells. Keeping the total amount of Kv2.1 plasmid constant, we found a significant decrease in the magnitude of currents with increasing Kv6.4 dose (ANOVA, p<0.05). To more cleanly assess biophysical differences between homo- and heterotetramers, we generated a Kv2.1-Kv6.4 dimer and compared currents recorded from cells transfected with Kv2.1 plasmid only vs those transfected with dimer plasmid only. We found a significant hyperpolarizing shift in inactivation V50 (−79.6 mV vs −53.5 mV, t-test, p=0.0001), significantly larger activation slope (20.7 vs 14.9, t-test, p=0.0014) and faster activation kinetics (2-way ANOVA, p=0.0354) for the dimer currents compared to Kv2.1-only currents. These results highlight several ways in which silent subunits modify the function of Kv2.1 currents.

Genome organization controls transcriptional dynamics during development.

Past studies offer contradictory claims for the role of genome organization in the regulation of gene activity. Here, we show through high-resolution chromosome conformation analysis that the Drosophila genome is organized by two independent classes of regulatory sequences, tethering elements and insulators. Quantitative live imaging and targeted genome editing demonstrate that this two-tiered organization is critical for the precise temporal dynamics of Hox gene transcription during development. Tethering elements mediate long-range enhancer-promoter interactions and foster fast activation kinetics. Conversely, the boundaries of topologically associating domains (TADs) prevent spurious interactions with enhancers and silencers located in neighboring TADs. These two levels of genome organization operate independently of one another to ensure precision of transcriptional dynamics and the reliability of complex patterning processes.

Magnesium Alloys for Hydrogen Storage Processed by ECAP Followed by Low Temperature Rolling

The hydrogen storage properties of pure magnesium and magnesium ZK60 alloy containing 2.5 wt% of mischmetal (Mm) processed by Cold Rolling (CR) and Low Temperature Rolling (LTR) were investigated. Before CR and LTR processing, Equal Channel Angular Pressing (ECAP) was employed in the alloys as an initial processing step to refine their microstructures. Microstructure findings showed that the ECAP+LTR route is more effective than the others to generate grain refinement - a desirable aspect in hydrogen storage materials. SEM and TEM results in combination with XPS analysis revealed that the ZK60 + 2.5 wt. % Mm alloy after being processed by ECAP + LTR had more refined grains (with sizes < 1μm) and exhibited superior resistance to the formation of oxides and/or hydroxides when compared with the pure magnesium alloy. The refined microstructures of the ECAP+LTR samples combined with the presence of highly oriented grains along to the (002) plane and large amount of defects – which are features associated to the LTR route - such as cracks and voids, resulted in a fast activation kinetics than for the samples processed by ECAP + CR route.

Cross Pharmacological, Biochemical and Computational Studies of a Human Kv3.1b Inhibitor from Androctonus australis Venom

The voltage-gated K+ channels Kv3.1 display fast activation and deactivation kinetics and are known to have a crucial contribution to the fast-spiking phenotype of certain neurons. AahG50, as a natural product extracted from Androctonus australis hector venom, inhibits selectively Kv3.1 channels. In the present study, we focused on the biochemical and pharmacological characterization of the component in AahG50 scorpion venom that potently and selectively blocks the Kv3.1 channels. We used a combined optimization through advanced biochemical purification and patch-clamp screening steps to characterize the peptide in AahG50 active on Kv3.1 channels. We described the inhibitory effect of a toxin on Kv3.1 unitary current in black lipid bilayers. In silico, docking experiments are used to study the molecular details of the binding. We identified the first scorpion venom peptide inhibiting Kv3.1 current at 170 nM. This toxin is the alpha-KTx 15.1, which occludes the Kv3.1 channel pore by means of the lysine 27 lateral chain. This study highlights, for the first time, the modulation of the Kv3.1 by alpha-KTx 15.1, which could be an interesting starting compound for developing therapeutic biomolecules against Kv3.1-associated diseases.

Heterogeneity and Remodeling of Ion Currents in Cultured Right Atrial Fibroblasts From Patients With Sinus Rhythm or Atrial Fibrillation.

Cardiac fibroblasts express multiple voltage-dependent ion channels. Even though fibroblasts do not generate action potentials, they may influence cardiac electrophysiology by electrical coupling via gap junctions with cardiomyocytes, and through fibrosis. Here, we investigate the electrophysiological phenotype of cultured fibroblasts from right atrial appendage tissue of patients with sinus rhythm (SR) or atrial fibrillation (AF). Using the patch-clamp technique in whole-cell mode, we observed steady-state outward currents exhibiting either no rectification or inward and/or outward rectification. The distributions of current patterns between fibroblasts from SR and AF patients were not significantly different. In response to depolarizing voltage pulses, we measured transient outward currents with fast and slow activation kinetics, an outward background current, and an inward current with a potential-dependence resembling that of L-type Ca2+ channels. In cell-attached patch-clamp mode, large amplitude, paxilline-sensitive single channel openings were found in ≈65% of SR and ∼38% of AF fibroblasts, suggesting the presence of “big conductance Ca2+-activated K+ (BKCa)” channels. The open probability of BKCa was significantly lower in AF than in SR fibroblasts. When cultured in the presence of paxilline, the shape of fibroblasts became wider and less spindle-like. Our data confirm previous findings on cardiac fibroblast electrophysiology and extend them by illustrating differential channel expression in human atrial fibroblasts from SR and AF tissue.

Low-Resistance silver bromide electrodes for recording fast ion channel kinetics under voltage clamp conditions

BackgroundTwo-electrode voltage clamp is a widely used technique for studying ionic currents. However, fast activation kinetics of ion channels are disguised by the capacitive transient during voltage clamp of Xenopus oocytes. The limiting factors of clamp performance include, but are not limited to, amplifier gain, membrane capacitance, and micropipette resistance. Previous work has focused on increasing amplifier gain (e.g.; high performing two-electrode amplifiers) or reducing the membrane capacitance (e.g.; the cut-open technique). New methodThe use of an Ag-AgBr electrode with saturated KBr solution to reduce micropipette resistance. ResultsThe conductivity of 4 M KBr was 37 % higher compared to 3 M KCl and the micropipette resistance was reduced by 19 % when 4 M KBr was used, compared to the standard 3 M KCl solution. Micropipette resistances correlated positively with capacitive transient durations. Neither the current-voltage relationship of the voltage-gated sodium channel, Nav1.7, nor Xenopus oocyte stability were affected by bromide ions. Comparison with existing methodsThe de facto standard for two-electrode voltage clamp is 3 M KCl and Ag-AgCl electrodes, which are associated an unnecessarily high micropipette resistance. Elsewise, cut-open voltage clamp techniques are technically demanding and require manipulation of the intracellular environment. ConclusionsThe use of an Ag-AgBr electrode with saturated KBr as micropipette solution reduces the capacitive transient in two-electrode voltage clamp recordings. Moreover, the exchange of chloride against bromide ions does not seem to affect oocyte physiology and ion channel kinetics.

Modulation of Kv3.1/Kv3.2 promotes gamma oscillations by rescuing Aβ-induced desynchronization of fast-spiking interneuron firing in an AD mouse model in vitro.

Gamma oscillations (30-80Hz) are important for cognitive functions and depend on the synchronized activity of fast-spiking interneurons (FSN), which is crucial for network stability. Gamma oscillations are degraded in Alzheimer's disease (AD) patients exhibiting cognitive impairment, with the degree of cognitive decline correlating with the severity of gamma disruption in response to neurotoxic amyloid-beta peptide (Aβ). Small molecule compounds EX15 and RE01modulate Kv3.1/Kv3.2 potassium channels on FSN, resulting in faster activation kinetics and increased firing frequency, suggesting direct consequences for cognition-relevant gamma oscillations, particularly in a situation where network activity is pathologically compromised in the presence of neurotoxic Aβ. Using electrophysiological techniques in an in vitro AD model, we found a significant effect of EX15 and RE01 with respect to counteracting toxic Aβ effects on neuronal dynamics, advocating for targeting FSN activity to rescue cognitive performance from impairment caused by neurodegenerative triggers. Rhythmic electrical activity in neuronal networks such as gamma oscillations (30-80Hz) underlies cognitive functions such as sensory perception, attention and memory. Gamma oscillations are disrupted in Alzheimer's disease (AD) patients and animal AD models, with the severity of cognitive decline correlating with the degree of rhythm disruption. Misfolded amyloid-β peptide (Aβ) is assumed to be a key trigger of AD pathology and has been show to de-synchronize action potential firing in fast-spiking interneurons (FSN), which is crucial for entraining neuronal network activity into the gamma rhythm. The synchronizing activity of FSN therefore has become one of the most suitable targets to counteract disease-driven degradation of gamma oscillations and consequent cognitive decline. EX15 and RE01 are small-molecule compounds that modulate Kv3.1/Kv3.2 potassium channels, resulting in faster activation kinetics and increased FSN firing frequency. In the present study, we investigated the potential pro-cognitive effects of EX15 and RE01 by testing their ability to modulate FSN activity during ongoing gamma oscillations in normal and Aβ-disrupted network states in mouse hippocampus in vitro. In the compromised, but not the uncompromised, network state with gamma oscillations partially disrupted by Aβ, both compounds improve gamma oscillation regularity by promoting re-synchronization of FSN action potential firing. Our data suggest a therapeutic potential for compounds such as EX15 and RE01, which can rescue normal action potential firing parameters in FSN, in the search for disease-modifying drug candidates counteracting the progressive dysfunction of neuronal network dynamics that underlies the cognitive impairment typical of AD and other cognitive brain disorders.

A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells.

Light-regulated modules offer unprecedented new ways to control cellular behaviour with precise spatial and temporal resolution. Among a variety of bacterial light-switchable gene expression systems, single-component systems consisting of single transcription factors would be more useful due to the advantages of speed, simplicity, and versatility. In the present study, we developed a single-component light-activated bacterial gene expression system (eLightOn) based on a novel LOV domain from Rhodobacter sphaeroides (RsLOV). The eLightOn system showed significant improvements over the existing single-component bacterial light-activated expression systems, with benefits including a high ON/OFF ratio of >500-fold, a high activation level, fast activation kinetics, and/or good adaptability. Additionally, the induction characteristics, including regulatory windows, activation kinetics and light sensitivities, were highly tunable by altering the expression level of LexRO. We demonstrated the usefulness of the eLightOn system in regulating cell division and swimming by controlling the expression of the FtsZ and CheZ genes, respectively, as well as constructing synthetic Boolean logic gates using light and arabinose as the two inputs. Taken together, our data indicate that the eLightOn system is a robust and highly tunable tool for quantitative and spatiotemporal control of bacterial gene expression.

Effects of graphite addition and air exposure on ball-milled Mg–Al alloys for hydrogen storage

Magnesium hydride (MgH2) is a promising candidate as a hydrogen storage material. However, its hydrogenation kinetics and thermodynamic stability still have room for improvement. Alloying Mg with Al has been shown to reduce the heat of hydrogenation and improve air resistance, whereas graphite helps accelerating hydrogenation kinetics in pure Mg. In this study, the effects of simultaneous Al alloying and graphite addition on the kinetics and air-exposure resistance were investigated on the Mg60Al40 system. The alloys were pulverized through high-energy ball milling (hereinafter HEBM). We tested different conditions of milling energy, added graphite contents, and air exposure times. Structural characterization was conducted via X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM). H2 absorption and desorption properties were obtained through volumetry in a Sieverts-type apparatus and Differential Scanning Calorimetry (DSC). The desorption activation energies were calculated using DSC curves through Kissinger analysis. Mg60Al40 with 10 wt% graphite addition showed fast activation kinetics, even after 2 years of air exposure. Graphite addition provided a catalytic effect on ball-milled Mg–Al alloys by improving both absorption and desorption kinetics and lowering the activation energy for desorption from 189 kJ/mol to 134 kJ/mol. The fast kinetics, reduced heat of reaction, and improved air resistance of these materials make them interesting candidates for potential application in hydride-based hydrogen storage tanks.

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials

The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2 -interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.

Piezo2 Mechanosensitive Ion Channel Role in Primary Enterochromaffin (EC) Cell Mechanosensitivity

INTRODUCTIONIntestinal enterochromaffin (EC) cells are the specialized epithelial mechanosensors of the gastrointestinal (GI) epithelium and are functionally and developmentally like Merkel cells in the skin. EC cells release serotonin (5‐HT) in response to mechanical stimuli. Recently, we discovered that like Merkel cells, human and mouse EC cells express the mechanosensitive (MS) ion channel Piezo2. However, little is known about the functional properties of this channel in primary EC cells.AIMTo determine the role of Piezo2 in primary EC cells.METHODSThe enteroendocrine cell transcription factor NeuroD1 was used to mark the late stage of EC cell development and generate NeuroD1‐cre::GCaMP5/tdTomato mouse to identify EC cells and examine their Ca2+ dynamics. We examined colocalization of tdTomato with 5‐HT and Piezo2 by immunohistochemistry. We established and used primary colon epithelium cultures for whole‐cell patch clamp and Ca2+ imaging experiments and mechanically stimulated by membrane displacement with a piezoelectrically driven glass probe or shear flow.RESULTSImmunostaining showed no tdTomato labeling outside of the GI epithelium, and epithelial tdTomato cells were 5‐HT+ and Piezo2+. Mechanical stimulation of primary EC cells by membrane displacement produced inward MS currents (20.8±4.9 pA/pF, Cm=3.5 pF, n=6) with fast activation and inactivation kinetics (τinact 11.1±2.8 ms), steep mechanosensitivity (Boltzmann function fit with a mid‐point of 3.46±0.80 μm and slope 0.79±0.27 μm) and a linear current‐voltage relationship (slope 0.61±0.17 pA/mV, x‐intercept −4.26±1.34 mV, n=5). Shear flow (20 s) transiently increased intracellular calcium ((Ca2+)i) (2.15±0.5 ΔF/F0, upstroke 3–5 s, return to baseline in 60 s, n=12). MS currents were inhibited by the MS ion channel blocker Gd3+ (20.8±4.9 pA/pF versus 5.3±1.9 pA/pF), Piezo channel blocker GsMTx‐4 (20.8±4.9 pA/pF versus 2.5±0.9 pA/pF) and Piezo2 siRNA (20.8±4.9 pA/pF versus 1.1±0.3 pA/pF), but not non‐targeted (NT) siRNA (20.8±4.9 pA/pF versus 16.0±5.2 pA/pF and) (n=4–6 for each condition, p&lt;0.05 for all except NT siRNA). Similarly, the (Ca2+)i responses to force were inhibited by Gd3+ (2.28±0.7 ΔF/F0 to 0.09±0.05 ΔF/F0), GsMTx‐4 (2.0±0.6 ΔF/F0 to 0.3±0.1 ΔF/F0), Piezo2 siRNA (2.15±0.5 ΔF/F0 to 0.08±0.05 ΔF/F0), but not NT siRNA (2.15±0.5 ΔF/F0 to 2.7±0.3 ΔF/F0) (n=4–6, p&lt;0.05 for all except NT siRNA).CONCLUSIONSIn the mouse epithelium, Piezo2+ EC cells were specifically marked by NeuroD1. Primary colonic NeuroD1+ cells had inward and non‐rectifying MS currents that could be blocked by Gd3+, GsMTx‐4 and Piezo2 siRNA. Similarly, shear flow evoked changes in (Ca2+)i that were also blocked by Piezo2 inhibitors and dependent on extracellular Ca2+. These results suggest that the MS ion channel Piezo2 acts as the primary EC cell mechanosensor, whose activation by force leads to MS ionic current and (Ca2+)i increase that may mediate mechanically‐induced 5‐HT release.Support or Funding InformationThis work was supported by NIH K08 DK106456, AGA Research Scholar Award, Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567) and DK52766.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Nitric Oxide on Voltage-Gated K⁺ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation.

This study investigated the expression of voltage-gated K+ (KV) channels in human cardiac fibroblasts (HCFs), and the effect of nitric oxide (NO) on the KV currents, and the underlying phosphorylation mechanisms. In reverse transcription polymerase chain reaction, two types of KV channels were detected in HCFs: delayed rectifier K+ channel and transient outward K+ channel. In whole-cell patch-clamp technique, delayed rectifier K+ current (IK) exhibited fast activation and slow inactivation, while transient outward K+ current (Ito) showed fast activation and inactivation kinetics. Both currents were blocked by 4-aminopyridine. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), increased the amplitude of IK in a concentration-dependent manner with an EC50 value of 26.4 µM, but did not affect Ito. The stimulating effect of SNAP on IK was blocked by pretreatment with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or by KT5823. 8-bromo-cyclic GMP stimulated the IK. The stimulating effect of SNAP on IK was also blocked by pretreatment with KT5720 or by SQ22536. Forskolin and 8-bromo-cyclic AMP each stimulated IK. On the other hand, the stimulating effect of SNAP on IK was not blocked by pretreatment of N-ethylmaleimide or by DL-dithiothreitol. Our data suggest that NO enhances IK, but not Ito, among KV currents of HCFs, and the stimulating effect of NO on IK is through the PKG and PKA pathways, not through S-nitrosylation.

Calcium-activated BKCa channels govern dynamic membrane depolarizations of horizontal cells in rodent retina.

Large conductance, Ca2+ -activated K+ (BKCa ) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells. BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150mm K+ , single channels had a conductance close to 250pS, within the range of all known BKCa channels. In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells. Large conductance, calcium-activated potassium (BKCa ) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+ ] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between -30 and +60mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole-cell outward currents were maximal at +50mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100nm), iberiotoxin (10μm), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30μm). Single channel recordings revealed the conductance of the channels to be 244±11pS (n=17; symmetrical 150mm K+ ) with open probability being both voltage- and Ca2+ -dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells.

Regulation of a Coupled MARCKS-PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module.

Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively coupled. Cytoplasmic Ca2+ is a recently recognized component of the feedback loop at the leading edge where it stimulates phosphoinositide-3-kinase (PI3K) and the production of its product signaling lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). A previous study implicated Ca2+-activated protein kinase C (PKC) and the phosphatidylinositol 4,5-bisphosphate (PIP2) binding protein MARCKS as two important players in this signaling, because PKC phosphorylation of MARCKS releases free PIP2 that serves as the membrane binding target and substrate for PI3K. This study asks whether calmodulin (CaM), which is known to directly bind MARCKS, also stimulates PIP3 production by releasing free PIP2. Single-molecule fluorescence microscopy is used to quantify the surface density and enzyme activity of key protein components of the hypothesized Ca2+-CaM-MARCKS-PIP2-PI3K-PIP3 circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP2 headgroups, thereby releasing free PIP2 that recruits active PI3K to the membrane and serves as the substrate for the generation of PIP3. The resulting CaM-triggered activation of PI3K is complete in seconds and is much faster than PKC-triggered activation, which takes minutes. Overall, the available evidence implicates both PKC and CaM in the coupling of Ca2+ and PIP3 signals and suggests these two different pathways have slow and fast activation kinetics, respectively.

Dynamic control of gene regulatory logic by seemingly redundant transcription factors.

Many transcription factors co-express with their homologs to regulate identical target genes, however the advantages of such redundancies remain elusive. Using single-cell imaging and microfluidics, we study the yeast general stress response transcription factor Msn2 and its seemingly redundant homolog Msn4. We find that gene regulation by these two factors is analogous to logic gate systems. Target genes with fast activation kinetics can be fully induced by either factor, behaving as an 'OR' gate. In contrast, target genes with slow activation kinetics behave as an 'AND' gate, requiring distinct contributions from both factors, upon transient stimulation. Furthermore, such genes become an 'OR' gate when the input duration is prolonged, suggesting that the logic gate scheme is not static but rather dependent on the input dynamics. Therefore, Msn2 and Msn4 enable a time-based mode of combinatorial gene regulation that might be applicable to homologous transcription factors in other organisms.

Activation Mechanism of the Bacteroides fragilis Cysteine Peptidase, Fragipain.

Enterotoxigenic Bacteroides fragilis produces a secreted metalloprotease known as B. fragilis toxin (BFT), which contributes to anaerobic sepsis, colitis, and colonic malignancy in mouse models of disease. A C11 family cysteine protease, fragipain (Fpn), directly activates BFT in the B. fragilis cell by removing the BFT prodomain. Fpn is itself a proenzyme and is autoactivated upon cleavage at an arginine residue in its activation loop. We have defined the proteolytic active site of Fpn, demonstrated that Fpn autoactivation can occur by an in trans loop cleavage mechanism, and characterized structural features of the Fpn activation loop that control peptidase activity against several substrates, including BFT. An arginine residue at the autocleavage site determines the fast activation kinetics of Fpn relative to the homologous C11 protease, PmC11, which is cleaved at lysine. Arginine to alanine substitution at the cleavage site ablated peptidase activity, as did partial truncation of the Fpn activation loop. However, complete truncation of the activation loop yielded an uncleaved, pro form of Fpn that was active as a peptidase against both Fpn and BFT substrates. Thus, Fpn can be transformed into an active peptidase in the absence of activation loop cleavage. This study provides insight into the mechanism of fragipain activation and, more generally, defines the role of the C11 activation loop in the control of peptidase activity and substrate specificity.

Cholesterol influences potassium currents in inner hair cells isolated from guinea pig cochlea.

There is a correlation between serum hyperlipidemia and hearing loss. Cholesterol is an integral component of the cell membrane and regulates the activity of ion channels in the lipid bilayer. The aim of this study was to investigate the effects of cholesterol on the potassium currents in IHCs by using the cholesterol-depleting drug, MβCD, and water-soluble cholesterol. IHCs were acutely isolated from a mature guinea-pig cochlea and potassium currents were recorded. MβCD and water-soluble cholesterol were applied to IHCs under pressure puff pipettes. IHCs showed outwardly rectifying currents (IK,f and IK,s) in response to depolarizing voltage pulses, with only a slight inward current (IK,n) when hyperpolarized. In 10mM MβCD solutions, the amplitude of outward K currents reversely decreased; however, fast activation kinetics was preserved. In contrast, in solution of 1mM water-soluble cholesterol, the amplitude of outward K currents reversely increased. At the membrane potential of +110mV, relative conductances were 0.87±0.07 and 1.18±0.11 in MβCD solutions and cholesterol solutions, respectively. The amplitude of K currents in isolated IHCs was reversely changed by cholesterol-depleting drug and water-soluble cholesterol. These results demonstrated the possibility of the involvement of IHC function in hyperlipidemia-induced inner ear disorders.

TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Structural Basis for Differences in Dynamics Induced by Leu Versus Ile Residues in the CD Loop of Kir Channels.

The effect of the conserved Leu/Ile site in the CD loop on the gating dynamics of Kir channels and corresponding micro-structural mechanism remains unclear. Molecular dynamics simulations were performed to investigate the structural mechanism of chicken Kir2.2. Compared to WT, the I223L mutant channel bound to PIP2 more strongly, was activated more rapidly, and maintained the activation state more stably after PIP2 dissociation. Cellular electrophysiology assays of mouse Kir2.1 and human Kir2.2 indicated that, consistent with simulations, the Leu residue increased the channel responses to PIP2 through increased binding affinity and faster activation kinetics, and the deactivation kinetics decreased upon PIP2 inhibition. The Ile residue induced the opposite responses. This difference was attributed to the distinct hydrophobic side chain symmetries of Leu and Ile; switching between these residues caused the interaction network to redistribute and offered effective conformation transduction in the Leu systems, which had more rigid and independent subunits.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9466-x) contains supplementary material, which is available to authorized users.