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Ion Channel Activity (ion + channel_activity)
Selected AbstractsIon channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporterBIOPOLYMERS, Issue 8 2010uková Abstract Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure,function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His211 were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure,function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718,726, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Photosensory Functions of Channelrhodopsins in Native Algal Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2009Oleg A. Sineshchekov Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light-gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin-2) is biochemical activation of secondary Ca2+ -channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells. [source] Isolated plant nuclei as mechanical and thermal sensors involved in calcium signallingTHE PLANT JOURNAL, Issue 1 2004Tou Cheu Xiong Summary Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus. [source] 3133: Planar patch-clamping in human corneal endothelial cells: a new tool for clinical application?ACTA OPHTHALMOLOGICA, Issue 2010S MERGLER Purpose Identification of apoptotic or damaged human corneal endothelial cells (HCECs) is limited to morphological evaluation such as phase contrast microscopy and vital staining. The molecular mechanisms of corneal endothelial cell loss are not fully understood. Special investigations in cellular signalling and ion channel research are necessary to elucidate the mechanisms of corneal cell loss. In this context, it is known that this cell loss is often caused by apoptosis in oxidative stress. Methods Automated planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. A particularly successful automated approach is based on planar patch-clamp chips and this is the basis for the technology used here. Routine intracellular or extracellular perfusion opens possibilities for studying the regulation and pharmacology of ion channels. Previously, these studies were available only to highly skilled and dedicated experimenters. Results Notable, definite ion channel activities could be demonstrated by conventional as well as by planar patch-clamp in HCECs for the first time. In particular, temperature-sensing transient receptor potential (TRP)-like non-selective cation channel currents as well as capsaicin-sensitive ion channel currents could be detected. The expression of TRPV1-3 ion channels in HCEC could also be confirmed by RT-PCR, Western blot analysis and fluorescence cell imaging. Conclusion The administration of this novel measuring technology opens new perspectives in the investigation of the physiology of HCEC. The findings may have direct clinical implication (eye banking procedures, keratoplasty). [source] Cysteine-rich secretory proteins are not exclusively expressed in the male reproductive tractDEVELOPMENTAL DYNAMICS, Issue 11 2008Thulasimala Reddy The Cysteine-RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen-5 proteins and the Pathogenesis related-1 (Pr-1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology. Developmental Dynamics 237:3313,3323, 2008. © 2008 Wiley-Liss, Inc. [source] Antiepileptic Drugs: How They Work in HeadacheHEADACHE, Issue 2001F. Michael Cutrer MD Antiepileptic drugs (AEDs) are promising agents for the prevention of migraine and other head pain. Migraine and epilepsy share several clinical features and respond to many of the same pharmacologic agents, suggesting that similar mechanisms may be involved in their pathophysiology. The mechanisms of action of AEDs are not fully understood, and a single drug may have more than one mechanism, both in epilepsy and in migraine. Valproate, topiramate, and gabapentin are likely to affect nociception by modulating gamma-aminobutyric acid- (GABA-) and/or glutamate-mediated neurotransmission. All three AEDs enhance GABA-mediated inhibition. Valproate and gabapentin interfere with GABA metabolism to prevent its ultimate conversion to succinate, and topiramate potentiates GABA-mediated inhibition by facilitating the action of GABA receptors. In addition, topiramate acts directly on non- N -methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate glutamate receptors. Valproate, topiramate, and possibly gabapentin inhibit sodium ion channels. All three drugs modulate calcium ion channel activity. Valproate blocks T-type calcium ion channels; topiramate inhibits high-voltage-activated L-type calcium ion channels; and gabapentin binds to the ,2, subunit of L-type calcium ion channels. AEDs may be useful in migraine prevention through such mechanisms as modulating the biochemical phenomena of aura or acting directly on the nociceptive system. Further evaluations of AEDs in migraine models will provide a better understanding of the pathophysiology and prevention of migraine. [source] Actions of Acute and Chronic Ethanol on Presynaptic TerminalsALCOHOLISM, Issue 2 2006Marisa Roberto This article presents the proceedings of a symposium entitled "The Tipsy Terminal: Presynaptic Effects of Ethanol" (held at the annual meeting of the Research Society on Alcoholism, in Santa Barbara, CA, June 27, 2005). The objective of this symposium was to focus on a cellular site of ethanol action underrepresented in the alcohol literature, but quickly becoming a "hot" topic. The chairs of the session were Marisa Roberto and George Robert Siggins. Our speakers were chosen on the basis of the diverse electrophysiological and other methods used to discern the effects of acute and chronic ethanol on presynaptic terminals and on the basis of significant insights that their data provide for understanding ethanol actions on neurons in general, as mechanisms underlying problematic behavioral effects of alcohol. The 5 presenters drew from their recent studies examining the effects of acute and chronic ethanol using a range of sophisticated methods from electrophysiological analysis of paired-pulse facilitation and spontaneous and miniature synaptic currents (Drs. Weiner, Valenzuela, Zhu, and Morrisett), to direct recording of ion channel activity and peptide release from acutely isolated synaptic terminals (Dr. Treistman), to direct microscopic observation of vesicular release (Dr. Morrisett). They showed that ethanol administration could both increase and decrease the probability of release of different transmitters from synaptic terminals. The effects of ethanol on synaptic terminals could often be correlated with important behavioral or developmental actions of alcohol. These and other novel findings suggest that future analyses of synaptic effects of ethanol should attempt to ascertain, in multiple brain regions, the role of presynaptic terminals, relevant presynaptic receptors and signal transduction linkages, exocytotic mechanisms, and their involvement in alcohol's behavioral actions. Such studies could lead to new treatment strategies for alcohol intoxication, alcohol abuse, and alcoholism. [source] Nitric oxide synthesis and signalling in plantsPLANT CELL & ENVIRONMENT, Issue 5 2008IAN D. WILSON ABSTRACT As with all organisms, plants must respond to a plethora of external environmental cues. Individual plant cells must also perceive and respond to a wide range of internal signals. It is now well-accepted that nitric oxide (NO) is a component of the repertoire of signals that a plant uses to both thrive and survive. Recent experimental data have shown, or at least implicated, the involvement of NO in reproductive processes, control of development and in the regulation of physiological responses such as stomatal closure. However, although studies concerning NO synthesis and signalling in animals are well-advanced, in plants there are still fundamental questions concerning how NO is produced and used that need to be answered. For example, there is a range of potential NO-generating enzymes in plants, but no obvious plant nitric oxide synthase (NOS) homolog has yet been identified. Some studies have shown the importance of NOS-like enzymes in mediating NO responses in plants, while other studies suggest that the enzyme nitrate reductase (NR) is more important. Still, more published work suggests the involvement of completely different enzymes in plant NO synthesis. Similarly, it is not always clear how NO mediates its responses. Although it appears that in plants, as in animals, NO can lead to an increase in the signal cGMP which leads to altered ion channel activity and gene expression, it is not understood how this actually occurs. NO is a relatively reactive compound, and it is not always easy to study. Furthermore, its biological activity needs to be considered in conjunction with that of other compounds such as reactive oxygen species (ROS) which can have a profound effect on both its accumulation and function. In this paper, we will review the present understanding of how NO is produced in plants, how it is removed when its signal is no longer required and how it may be both perceived and acted upon. [source] Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1PROTEIN SCIENCE, Issue 3 2002Che Ma HIV-1, human immunodeficiency virus type 1; AIDS, acquired immune deficiency syndrome; NMR, nuclear magnetic resonance; CNBr, cyanogen bromide; DHPC, dihexanoyl phosphatidylcholine; TROSY, transverse relaxation-optimized spectroscopy Abstract Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2,81). Vpu2,37 encompasses the N-terminal transmembrane ,-helix; Vpu2,51 spans the N-terminal transmembrane helix and the first cytoplasmic ,-helix; Vpu28,81 includes the entire cytoplasmic domain containing the two C-terminal amphipathic ,-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane ,-helix. The C-terminal ,-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation. [source] A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neuronsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Christophe Py Abstract We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593,600. © 2010 Wiley Periodicals, Inc. [source] Using gene chips to identify organ-specific, smooth muscle responses to experimental diabetes: potential applications to urological diseasesBJU INTERNATIONAL, Issue 2 2007Jason D. Hipp OBJECTIVE To identify early diabetes-related alterations in gene expression in bladder and erectile tissue that would provide novel diagnostic and therapeutic treatment targets to prevent, delay or ameliorate the ensuing bladder and erectile dysfunction. MATERIALS AND METHODS The RG-U34A rat GeneChip® (Affymetrix Inc., Sunnyvale, CA, USA) oligonucleotide microarray (containing ,8799 genes) was used to evaluate gene expression in corporal and male bladder tissue excised from rats 1 week after confirmation of a diabetic state, but before demonstrable changes in organ function in vivo. A conservative analytical approach was used to detect alterations in gene expression, and gene ontology (GO) classifications were used to identify biological themes/pathways involved in the aetiology of the organ dysfunction. RESULTS In all, 320 and 313 genes were differentially expressed in bladder and corporal tissue, respectively. GO analysis in bladder tissue showed prominent increases in biological pathways involved in cell proliferation, metabolism, actin cytoskeleton and myosin, as well as decreases in cell motility, and regulation of muscle contraction. GO analysis in corpora showed increases in pathways related to ion channel transport and ion channel activity, while there were decreases in collagen I and actin genes. CONCLUSIONS The changes in gene expression in these initial experiments are consistent with the pathophysiological characteristics of the bladder and erectile dysfunction seen later in the diabetic disease process. Thus, the observed changes in gene expression might be harbingers or biomarkers of impending organ dysfunction, and could provide useful diagnostic and therapeutic targets for a variety of progressive urological diseases/conditions (i.e. lower urinary tract symptoms related to benign prostatic hyperplasia, erectile dysfunction, etc.). [source] Oscillatory transient inward currents in ventricular myocytes of healthy versus myopathic Syrian hamsterCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 10 2004Sze-Hsueh Wu Summary 1.,The present experiments were performed in order to study abnormal action potential configuration and ion channel activity in ventricular myocytes obtained from 23 male myopathic Syrian hamsters (Biobreeders strain 14.6, 32,52 weeks old) compared with 10 age-matched healthy control hamsters (Biobreeders F1B) by means of whole-cell patch-clamp techniques. 2.,The results show that the myopathic myocytes had a longer action potential duration, a reduced transient outward K+ current on depolarization and a smaller transient inward current on repolarization after prolonged depolarizing pulses (> 500 msec). However, the L-type Ca2+ current and the inwardly rectifing K+ current were not significantly different from those of healthy myocytes. 3.,The oscillatory transient inward currents could be diminished by treatment with ryanodine (0.01,1 µmol/L), a sarcoplasmic reticulum (SR) Ca2+ release channel blocker, or with Na+ -free superfusate. 4.,We conclude that the hereditary myopathic hamsters are less likely to develop delayed afterdepolarization-related transient inward currents and triggered arrhythmias owing to a smaller SR Ca2+ content. [source] | ||||||||||||||||