Journal articles on the topic 'Single-ion channel'
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1
Plested, Andrew J. R. "Single-Channel Recording of Ligand-Gated Ion Channels." Cold Spring Harbor Protocols 2016, no. 8 (August 2016): pdb.top087239. http://dx.doi.org/10.1101/pdb.top087239.
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Mortensen, Martin, and Trevor G. Smart. "Single-channel recording of ligand-gated ion channels." Nature Protocols 2, no. 11 (November 2007): 2826–41. http://dx.doi.org/10.1038/nprot.2007.403.
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JANG, J. H., S. D. KIM, J. B. PARK, S. J. HONG, and P. D. RYU. "Ion channels ofFasciola hepaticaincorporated into planar lipid bilayers." Parasitology 128, no. 1 (January 2004): 83–89. http://dx.doi.org/10.1017/s0031182003004232.
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Ion channels are important target sites of anthelmintics, but little is known about those inFasciola hepatica. In this work, we applied a planar lipid bilayer technique to characterize the properties of single ion channels inF. hepatica. Under a 200/40 mMKCl gradient, a large conductance channel of 251 pS was observed in 18% of the membranes studied. The channel was selective to K+over Cl−with a permeability ratio of K+to Cl−(PK/PCl) of 4·9. Open state probability (Po) of the channel was less than 0·5 and dependent on voltage (−60~+40 mV) and Ca2+(~100 μM). The other two types of single channels observed in 11 and 5% of membranes, respectively, were a K+-permeable channel of 80 pS (PK/PCl=4·6) and a Cl−-permeable channel of 64 pS (PK/PCl=0·058). Open state probability of both channels showed little voltage dependence. The results indicate that distinct single channels of 60~251 pS are present in relative abundance and, in addition, that the planar lipid bilayer technique can be a useful tool for the study of single ion channels inF. hepatica.
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Baker, Mariah R., Guizhen Fan, and Irina I. Serysheva. "Single-particle cryo-EM of the ryanodine receptor channel." European Journal of Translational Myology 25, no. 1 (January 12, 2015): 35. http://dx.doi.org/10.4081/ejtm.2015.4803.
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Ryanodine receptors (RyRs) are tetrameric ligand-gated Ca2+ release channels that are responsible for the increase of cytosolic Ca2+ concentration leading to muscle contraction. Our current understanding of RyR channel gating and regulation is greatly limited due to the lack of a high-resolution structure of the channel protein. The enormous size and unwieldy shape of Ca2+ release channels make X-ray or NMR methods difficult to apply for high-resolution structural analysis of the full-length functional channel. Single-particle electron cryo-microscopy (cryo-EM) is one of the only effective techniques for the study of such a large integral membrane protein and its molecular interactions. Despite recent developments in cryo-EM technologies and break-through single-particle cryo-EM studies of ion channels, cryospecimen preparation, particularly the presence of detergent in the buffer, remains the main impediment to obtaining atomic-resolution structures of ion channels and a multitude of other integral membrane protein complexes. In this review we will discuss properties of several detergents that have been successfully utilized in cryo-EM studies of ion channels and the emergence of the detergent alternative amphipol to stabilize ion channels for structure-function characterization. Future structural studies of challenging specimen like ion channels are likely to be facilitated by cryo-EM amenable detergents or alternative surfactants.
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Fox, J. A., B. A. Pfeffer, and G. L. Fain. "Single-channel recordings from cultured human retinal pigment epithelial cells." Journal of General Physiology 91, no. 2 (February 1, 1988): 193–222. http://dx.doi.org/10.1085/jgp.91.2.193.
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We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.
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Rodriguez-Contreras, Adrian, Ping Lv, Jun Zhu, Hyo Jeong Kim, and Ebenezer N. Yamoah. "Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells." Journal of Neurophysiology 100, no. 4 (October 2008): 2115–24. http://dx.doi.org/10.1152/jn.90473.2008.
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To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.
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Oh, Y., and D. J. Benos. "Single-channel characteristics of a purified bovine renal amiloride-sensitive Na+ channel in planar lipid bilayers." American Journal of Physiology-Cell Physiology 264, no. 6 (June 1, 1993): C1489—C1499. http://dx.doi.org/10.1152/ajpcell.1993.264.6.c1489.
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We have purified an amiloride-inhibitable Na+ channel protein from bovine renal papillae using ion-exchange and immunoaffinity chromatography. In the present study, these purified Na+ channels were reconstituted into planar lipid bilayers, and their single-channel characteristics were studied. We observed both large- and small-conductance Na(+)-selective ion channels in planar lipid bilayers. Single-channel conductance for the large- and small-conductance channels saturated as a function of Na+ concentration. These relations could be fitted by a simple Langmuir isotherm with a Michaelis constant of 55 and 45 mM and a maximum open-state conductance of 56 or 8.4 pS, respectively. Both channels were perfectly cation selective, with a Na(+)-to-K+ permeability ratio of 6.7:1 for the large channel and 7.8:1 for the small channel, and their open single-channel current-voltage relations were linear when bathed with symmetrical Na+ solutions. The percent open time of the reconstituted large or small channels varied between 10 and 50% or 1 and 20%, respectively. After application of amiloride, both the large- and small-conductance Na+ channels were inhibited in a dose-dependent manner.
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Lozanović Šajić, Jasmina, Sonja Langthaler, and Christian Baumgartner. "Creating a Novel Mathematical Model of the Kv10.1 Ion Channel and Controlling Channel Activity with Nanoelectromechanical Systems." Applied Sciences 12, no. 8 (April 11, 2022): 3836. http://dx.doi.org/10.3390/app12083836.
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The use of nanoelectromechanical systems or nanorobots offers a new concept for sensing and controlling subcellular structures, such as ion channels. We present here a novel method for mathematical modeling of ion channels based on control system theory and system identification. We investigated the use of nanoelectromechanical devices to control the activity of ion channels, particularly the activity of the voltage-gated ion channel Kv10.1, an important channel in cancer development and progression. A mathematical model of the dynamic behavior of the selected ion channel Kv10.1 in the Laplace (s) domain was developed, which is given in the representation of a transfer function. In addition, we addressed the possibilities of controlling ion channel activity by nanoelectromechanical devices and nanorobots and finally presented a control algorithm for the Kv10.1 as a control object. A use case demonstrates the potential of a Kv10.1 controlled nanorobot for cancer treatment at a single-cell level.
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Gao, Jianzhao, Hong Wei, Alberto Cano, and Lukasz Kurgan. "PSIONplusm Server for Accurate Multi-Label Prediction of Ion Channels and Their Types." Biomolecules 10, no. 6 (June 7, 2020): 876. http://dx.doi.org/10.3390/biom10060876.
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Computational prediction of ion channels facilitates the identification of putative ion channels from protein sequences. Several predictors of ion channels and their types were developed in the last quindecennial. While they offer reasonably accurate predictions, they also suffer a few shortcomings including lack of availability, parallel prediction mode, single-label prediction (inability to predict multiple channel subtypes), and incomplete scope (inability to predict subtypes of the voltage-gated channels). We developed a first-of-its-kind PSIONplusm method that performs sequential multi-label prediction of ion channels and their subtypes for both voltage-gated and ligand-gated channels. PSIONplusm sequentially combines the outputs produced by three support vector machine-based models from the PSIONplus predictor and is available as a webserver. Empirical tests show that PSIONplusm outperforms current methods for the multi-label prediction of the ion channel subtypes. This includes the existing single-label methods that are available to the users, a naïve multi-label predictor that combines results produced by multiple single-label methods, and methods that make predictions based on sequence alignment and domain annotations. We also found that the current methods (including PSIONplusm) fail to accurately predict a few of the least frequently occurring ion channel subtypes. Thus, new predictors should be developed when a larger quantity of annotated ion channels will be available to train predictive models.
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Lu, Tao, Li Wu, Jun Xiao, and Jian Yang. "Permeant Ion-Dependent Changes in Gating of Kir2.1 Inward Rectifier Potassium Channels." Journal of General Physiology 118, no. 5 (November 1, 2001): 509–22. http://dx.doi.org/10.1085/jgp.118.5.509.
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We studied the effect of monovalent thallium ion (Tl+) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K+ to Tl+ decreases the mean open-time by ∼20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (τ = 2.7 ms at −100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl+ must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl+ current. These results suggest that Tl+ ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K+ with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl+ greatly diminishes Ba2+ block. The unblocking rate of Ba2+ is increased by >22-fold when the external permeant ion is switched from K+ to Tl+ regardless of the direction of Ba2+ exit. This effect cannot be explained solely by ion–ion interactions, but is consistent with the notion that Tl+ induces conformational changes in the selectivity filter.
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Ranatunga, Kishani M., Charlotte Adcock, Ian D. Kerr, Graham R. Smith, and Mark S. P. Sansom. "Ion channels of biological membranes: prediction of single channel conductance." Theoretical Chemistry Accounts: Theory, Computation, and Modeling (Theoretica Chimica Acta) 101, no. 1-3 (February 15, 1999): 97–102. http://dx.doi.org/10.1007/s002140050414.
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ROBERTSON, A. P., R. J. MARTIN, and J. R. KUSEL. "A vesicle preparation for resolving single-channel currents in tegument of male Schistosoma mansoni." Parasitology 115, no. 2 (August 1997): 183–92. http://dx.doi.org/10.1017/s0031182097001273.
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A tegumental vesicle preparation from adult male Schistosoma mansoni was developed that allows the resolution of single ion-channel currents. Adult male schistosomes were exposed to a low pH (3·75) medium for a period of approximately 30 min at 37°C. During this period smooth vesicles formed from the tegument. Fluorescence microscopy following staining of the tegument with the dye, 5-N-[octadecanoyl]aminofluorescein (AF-18), transmission electron microscopy and scanning electron microscopy revealed that the vesicles were produced from the outer tegumental membrane. The fluorescence studies showed the presence of the double bilayer structure of the outer membrane in >41% of the vesicles. These studies suggested that the preparation is suitable for single-channel recording with the patch-clamp technique. Cell-attached and isolated inside-out patch recordings of ion-channel activity were obtained with giga-ohm resistance seals. Different types of ion-channel were recorded from tegumental vesicles from male schistosomes, illustrating the potential of the technique. The channels observed included: a non-selective cation channel (360 pS); a K+ channel (with a conductance of 115 pS in high bath-K conditions); and a Cl− selective channel (20 pS). The currents of these ion-channels may cross the double bilayer of the outer tegumental membrane.
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Takumi, T. "A Protein With a Single Transmembrane Domain Forms an Ion Channel." Physiology 8, no. 4 (August 1, 1993): 175–78. http://dx.doi.org/10.1152/physiologyonline.1993.8.4.175.
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Isk is a small membrane protein with a single membrane-spanning domain and shows a slow voltage-dependent K+ channel activity. Mutational analyses showed that Isk forms an integral part of the K+ channel itself. Two other proteins have similar properties, suggesting a new group of voltage-dependent channels.
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Marabelli, Alessandro, Remigijus Lape, and Lucia Sivilotti. "Mechanism of activation of the prokaryotic channel ELIC by propylamine: A single-channel study." Journal of General Physiology 145, no. 1 (December 29, 2014): 23–45. http://dx.doi.org/10.1085/jgp.201411234.
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Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5–50 mM) in outside-out patches at −60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50–600 ms, 1–50 mM, −100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the “Flip” model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The “Primed” model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼102 (∼20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.
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Worden, M. K., R. Rahamimoff, and E. A. Kravitz. "Ion channel activity in lobster skeletal muscle membrane." Journal of Experimental Biology 182, no. 1 (September 1, 1993): 113–30. http://dx.doi.org/10.1242/jeb.182.1.113.
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Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32–380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.
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Fernández, José A., Roman Skryma, Gabriel Bidaux, Karl L. Magleby, C. Norman Scholfield, J. Graham McGeown, Natalia Prevarskaya, and Alexander V. Zholos. "Voltage- and cold-dependent gating of single TRPM8 ion channels." Journal of General Physiology 137, no. 2 (January 31, 2011): 173–95. http://dx.doi.org/10.1085/jgp.201010498.
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Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30°C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (Po) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of Po, the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation.
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Hirano, Minako, Daiki Yamamoto, Mami Asakura, Tohru Hayakawa, Shintaro Mise, Akinobu Matsumoto, and Toru Ide. "A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings." Micromachines 11, no. 12 (December 1, 2020): 1070. http://dx.doi.org/10.3390/mi11121070.
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Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, α-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.
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Korman, Can E., and Isaak D. Mayergoyz. "On hysteresis of ion channels." Mathematical Modelling of Natural Phenomena 15 (2020): 26. http://dx.doi.org/10.1051/mmnp/2019058.
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Ion channel proteins have many conformational (metastable) states and, for this reason, they exhibit hysteresis. This fact is responsible for the non-Markovian stochastic nature of single ion channel recordings. It is suggested in the paper that the stochastic single channel recordings can be modeled as the random outputs of rectangular hysteresis loops driven by stochastic processes. The latter problem can be mathematically treated as an exit problem for stochastic processes or by using the theory of stochastic processes on graphs. It is also demonstrated in the paper that the collective action of sodium and potassium channels responsible for the generation and propagation of action potentials exhibit hysteresis. This demonstration is accomplished by using the inverse problem approach to the nonlinear Hodgkin-Huxley diffusion equation.
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Shi, Wenqing, Yuhan Zeng, Lushan Zhou, Yucheng Xiao, Theodore R. Cummins, and Lane A. Baker. "Membrane patches as ion channel probes for scanning ion conductance microscopy." Faraday Discussions 193 (2016): 81–97. http://dx.doi.org/10.1039/c6fd00133e.
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We describe dual-barrel ion channel probes (ICPs), which consist of an open barrel and a barrel with a membrane patch directly excised from a donor cell. When incorporated with scanning ion conductance microscopy (SICM), the open barrel (SICM barrel) serves to measure the distance-dependent ion current for non-invasive imaging and positioning of the probe in the same fashion of traditional SICM. The second barrel with the membrane patch supports ion channels of interest and was used to investigate ion channel activities. To demonstrate robust probe control with the dual-barrel ICP-SICM probe and verify that the two barrels are independently addressable, current–distance characteristics (approach curves) were obtained with the SICM barrel and simultaneous, current–time (I–T) traces were recorded with the ICP barrel. To study the influence that the distance between ligand-gated ion channels (i.e., large conductance Ca2+-activated K+ channels/BK channels) and the ligand source (i.e., Ca2+ source) has on channel activations, ion channel activities were recorded at two fixed probe–substrate distances (Dps) with the ICP barrel. The two fixed positions were determined from approach curves acquired with the SICM barrel. One position was defined as the “In-control” position, where the probe was in close proximity to the ligand source; the second position was defined as the “Far” position, where the probe was retracted far away from the ligand source. Our results confirm that channel activities increased dramatically with respect to both open channel probability and single channel current when the probe was near the ligand source, as opposed to when the probe was far away from the ligand source.
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Erxleben, C. "Stretch-activated current through single ion channels in the abdominal stretch receptor organ of the crayfish." Journal of General Physiology 94, no. 6 (December 1, 1989): 1071–83. http://dx.doi.org/10.1085/jgp.94.6.1071.
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Single stretch-activated ion channels were studied on the soma and primary dendrites of stretch receptor neurons of the crayfish Orconectes limosus. When the membrane of the patch was deformed by applying suction to the pipette, a marked nonlinear increase in single-channel activity could be observed in two types of channels. These were indistinguishable on the basis of their single-channel conductances but differed in their voltage range of activation. One type showed strong inward rectification (RSA channel) and the second type was largely voltage independent (SA channel). A linear relationship was found between negative pressure and the natural logarithm of the channels' open probability. For an e-fold change in pressure, the average sensitivity was 8.7 +/- 0.4 (SD, n = 5) mmHg for the RSA channel and 5.6 +/- 2.2 (n = 5) mmHg for the SA channel. Both channels were found to be permeable to mono- and divalent cations. Current-voltage relationships were linear with slope conductances for the SA channel of: 71 +/- 11 (SD, n = 3) pS for K+, 50 +/- 7.4 (n = 5) pS for Na+, and 23 pS for Ca++. Similar values were found for the RSA channel. The data suggest that the SA channel is responsible for the mechanotransduction process in the stretch receptor neuron.
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Alvarez, Osvaldo, Carlos Gonzalez, and Ramon Latorre. "COUNTING CHANNELS: A TUTORIAL GUIDE ON ION CHANNEL FLUCTUATION ANALYSIS." Advances in Physiology Education 26, no. 4 (December 2002): 327–41. http://dx.doi.org/10.1152/advan.00006.2002.
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Ion channels open and close in a stochastic fashion, following the laws of probability. However, distinct from tossing a coin or a die, the probability of finding the channel closed or open is not a fixed number but can be modified (i.e., we can cheat) by some external stimulus, such as the voltage. Single-channel records can be obtained using the appropriate electrophysiological technique (e.g., patch clamp), and from these records the open probability and the channel conductance can be calculated. Gathering these parameters from a membrane containing many channels is not straightforward, as the macroscopic current I = iNPo, where i is the single-channel current, N the number of channels, and Po the probability of finding the channel open, cannot be split into its individual components. In this tutorial, using the probabilistic nature of ion channels, we discuss in detail how i, N, and Po max (the maximum open probability) can be obtained using fluctuation (nonstationary noise) analysis (Sigworth FJ. G Gen Physiol 307: 97–129, 1980). We also analyze the sources of possible artifacts in the determination of i and N, such as channel rundown, inadequate filtering, and limited resolution of digital data acquisition by use of a simulation computer program (available at www.cecs.cl ).
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Hess, P., J. B. Lansman, and R. W. Tsien. "Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells." Journal of General Physiology 88, no. 3 (September 1, 1986): 293–319. http://dx.doi.org/10.1085/jgp.88.3.293.
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Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.
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Ide, T., H. Sakamoto, and T. Yanagida. "Single molecule imaging of an ion-channel." Seibutsu Butsuri 40, supplement (2000): S205. http://dx.doi.org/10.2142/biophys.40.s205_4.
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Stava, E., Minrui Yu, Hyun Cheol Shin, and R. H. Blick. "Single-Ion Channel Recordings on Quartz Substrates." IEEE Transactions on NanoBioscience 9, no. 4 (December 2010): 307–9. http://dx.doi.org/10.1109/tnb.2010.2076830.
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Murray, Royce. "Single Ion Channel, Medicine and Measurement Science." Analytical Chemistry 63, no. 23 (December 1991): 1127a. http://dx.doi.org/10.1021/ac00023a600.
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Liu, Chang, Changan Xie, Khade Grant, Zhuocheng Su, Weihua Gao, Qinglian Liu, and Lei Zhou. "Patch-clamp fluorometry–based channel counting to determine HCN channel conductance." Journal of General Physiology 148, no. 1 (June 27, 2016): 65–76. http://dx.doi.org/10.1085/jgp.201511559.
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Counting ion channels on cell membranes is of fundamental importance for the study of channel biophysics. Channel counting has thus far been tackled by classical approaches, such as radioactive labeling of ion channels with blockers, gating current measurements, and nonstationary noise analysis. Here, we develop a counting method based on patch-clamp fluorometry (PCF), which enables simultaneous electrical and optical recordings, and apply it to EGFP-tagged, hyperpolarization-activated and cyclic nucleotide–regulated (HCN) channels. We use a well-characterized and homologous cyclic nucleotide–gated (CNG) channel to establish the relationship between macroscopic fluorescence intensity and the total number of channels. Subsequently, based on our estimate of the total number of HCN channels, we determine the single-channel conductance of HCN1 and HCN2 to be 0.46 and 1.71 pS, respectively. Such a small conductance would present a technical challenge for traditional electrophysiology. This PCF-based technique provides an alternative method for counting particles on cell membranes, which could be applied to biophysical studies of other membrane proteins.
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Guo, Rui, Weizhong Zeng, Hengjun Cui, Liping Chen, and Sheng Ye. "Ionic interactions of Ba2+ blockades in the MthK K+ channel." Journal of General Physiology 144, no. 2 (July 14, 2014): 193–200. http://dx.doi.org/10.1085/jgp.201411192.
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The movement and interaction of multiple ions passing through in single file underlie various fundamental K+ channel properties, from the effective conduction of K+ ions to channel blockade by Ba2+ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+ with permeant ions within the ion conduction pathway of the MthK K+ channel. We found that, as typical of K+ channels, the MthK channel was blocked by Ba2+ at the internal side, and the Ba2+-blocking effect was enhanced by external K+. We also obtained crystal structures of the MthK K+ channel pore in both Ba2+–Na+ and Ba2+–K+ environments. In the Ba2+–Na+ environment, we found that a single Ba2+ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba2+–K+ environment, Ba2+ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba2+ blockade of K+ channels.
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Sesti, Federico, and Steve A. N. Goldstein. "Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome." Journal of General Physiology 112, no. 6 (December 1, 1998): 651–63. http://dx.doi.org/10.1085/jgp.112.6.651.
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IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.
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Tavalin, Steven J., Dawn Shepherd, Robin K. Cloues, Sarah E. H. Bowden, and Neil V. Marrion. "Modulation of Single Channels Underlying Hippocampal L-Type Current Enhancement by Agonists Depends on the Permeant Ion." Journal of Neurophysiology 92, no. 2 (August 2004): 824–37. http://dx.doi.org/10.1152/jn.00700.2003.
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The influx of calcium (Ca2+) ions through L-type channels underlies many cellular processes, ranging from initiation of gene transcription to activation of Ca2+-activated potassium channels. L-type channels possess a diagnostic pharmacology, being enhanced by the dihydropyridine BAY K 8644 and benzoylpyrrole FPL 64176. It is assumed that the action of these compounds is independent of the ion conducted through the channel. In contrast to this assumption, modulation of L-type channel activity in acutely dissociated rat CA1 hippocampal neurons depended on the divalent ion identity. BAY K 8644 and FPL 64176 substantially increased single-channel open time only when barium (Ba2+) was the permeant ion. BAY K 8644 increased single-channel conductance when either Ba2+ or Ca2+ ions were the charge carrier, an effect not observed with FPL 64176. BAY K 8644 enhanced the whole cell L-type channel Ca2+- or Ba2+-carried current without a change in deactivation tail kinetics. In contrast, enhancement by FPL 64176 was associated with a dramatic slowing of deactivation kinetics only when Ba2+ and not Ca2+ was the charge carrier. Current activation was slowed by FPL 64176 with either charge carrier, an effect arising from a clustering of agonist-modified long-duration openings toward the end of the voltage step. These data indicate that agonists enhanced L-type current by distinct mechanisms dependent on the permeant ion, indicating that care must be considered when used as diagnostic tools.
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Murakami, Midori, and Hiromasa Kijima. "Transduction Ion Channels Directly Gated by Sugars on the Insect Taste Cell." Journal of General Physiology 115, no. 4 (April 1, 2000): 455–66. http://dx.doi.org/10.1085/jgp.115.4.455.
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Insects detect sugars and amino acids by a specialized taste cell, the sugar receptor cell, in the taste hairs located on their labela and tarsi. We patch-clamped sensory processes of taste cells regenerated from the cut end of the taste hairs on the labelum of the flashfly isolated from the pupa ∼20 h before emergence. We recorded both single channel and ensemble currents of novel ion channels located on the distal membrane of the sensory process of the sugar receptor cell. In the stable outside-out patch membrane excised from the sensory processes, we could repeatedly record sucrose-induced currents for tens of minutes without appreciable decrease. An inhibitor of G-protein activation, GDP-β-S, did not significantly decrease the sucrose response. These results strongly suggested that the channel is an ionotropic receptor (a receptor/channel complex), activated directly by sucrose without mediation by second messengers or G protein. The channel was shown to be a nonselective cation channel. Analyses of single channel currents showed that the sucrose-gated channel has a single channel conductance of ∼30 pS and has a very short mean open time of ∼0.23 ms. It is inhibited by external Ca2+ and the dose–current amplitude relation could be described by a Michaelis-Menten curve with an apparent dissociation constant of ∼270 mM. We also report transduction ion channels of the receptor/channel complex type directly gated by fructose and those gated by L-valine located on the sensory process.
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Kraske, Shane, J. Thomas Cunningham, George Hajduczok, Mark W. Chapleau, Francois M. Abboud, and Ruth E. Wachtel. "Mechanosensitive ion channels in putative aortic baroreceptor neurons." American Journal of Physiology-Heart and Circulatory Physiology 275, no. 4 (October 1, 1998): H1497—H1501. http://dx.doi.org/10.1152/ajpheart.1998.275.4.h1497.
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Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative aortic baroreceptor neurons were identified by labeling nerve endings in the adventitia of the aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of ∼0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 μM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with aortic endothelial cells, but not aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that aortic endothelial cells secrete a diffusible factor that increases channel expression.
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Goaillard, Jean-Marc, and Eve Marder. "Ion Channel Degeneracy, Variability, and Covariation in Neuron and Circuit Resilience." Annual Review of Neuroscience 44, no. 1 (July 8, 2021): 335–57. http://dx.doi.org/10.1146/annurev-neuro-092920-121538.
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The large number of ion channels found in all nervous systems poses fundamental questions concerning how the characteristic intrinsic properties of single neurons are determined by the specific subsets of channels they express. All neurons display many different ion channels with overlapping voltage- and time-dependent properties. We speculate that these overlapping properties promote resilience in neuronal function. Individual neurons of the same cell type show variability in ion channel conductance densities even though they can generate reliable and similar behavior. This complicates a simple assignment of function to any conductance and is associated with variable responses of neurons of the same cell type to perturbations, deletions, and pharmacological manipulation. Ion channel genes often show strong positively correlated expression, which may result from the molecular and developmental rules that determine which ion channels are expressed in a given cell type.
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Brachet, Anna, Christophe Leterrier, Marie Irondelle, Marie-Pierre Fache, Victor Racine, Jean-Baptiste Sibarita, Daniel Choquet, and Bénédicte Dargent. "Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier." Journal of Cell Biology 191, no. 2 (October 18, 2010): 383–95. http://dx.doi.org/10.1083/jcb.201003042.
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In mammalian neurons, the precise accumulation of sodium channels at the axonal initial segment (AIS) ensures action potential initiation. This accumulation precedes the immobilization of membrane proteins and lipids by a diffusion barrier at the AIS. Using single-particle tracking, we measured the mobility of a chimeric ion channel bearing the ankyrin-binding motif of the Nav1.2 sodium channel. We found that ankyrin G (ankG) limits membrane diffusion of ion channels when coexpressed in neuroblastoma cells. Site-directed mutants with decreased affinity for ankG exhibit increased diffusion speeds. In immature hippocampal neurons, we demonstrated that ion channel immobilization by ankG is regulated by protein kinase CK2 and occurs as soon as ankG accumulates at the AIS of elongating axons. Once the diffusion barrier is formed, ankG is still required to stabilize ion channels. In conclusion, our findings indicate that specific binding to ankG constitutes the initial step for Nav channel immobilization at the AIS membrane and precedes the establishment of the diffusion barrier.
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Haws, C. M., B. D. Winegar, and J. B. Lansman. "Block of single L-type Ca2+ channels in skeletal muscle fibers by aminoglycoside antibiotics." Journal of General Physiology 107, no. 3 (March 1, 1996): 421–32. http://dx.doi.org/10.1085/jgp.107.3.421.
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The activity of single L-type Ca2+ channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were concerned with the mechanism by which aminoglycoside antibiotics inhibit ion flow through the channel. Aminoglycosides produced discrete fluctuations in the single-channel current when added to the external solution. The blocking kinetics could be described as a simple bimolecular reaction between an aminoglycoside molecule and the open channel. The blocking rate was found to be increased when either the membrane potential was made more negative or the concentration of external permeant ion was reduced. Both of these effects are consistent with a blocking site that is located within the channel pore. Other features of block, however, were incompatible with a simple pore blocking mechanism. Hyperpolarization enhanced the rate of unblocking, even though an aminoglycoside molecule must dissociate from its binding site in the channel toward the external solution against the membrane field. Raising the external permeant ion concentration also enhanced the rate of unblocking. This latter finding suggests that aminglycoside affinity is modified by repulsive interactions that arise when the pore is simultaneously occupied by a permeant ion and an aminoglycoside molecule.
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Hedrich, Rainer. "Ion Channels in Plants." Physiological Reviews 92, no. 4 (October 2012): 1777–811. http://dx.doi.org/10.1152/physrev.00038.2011.
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Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K+-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K+ channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.
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Dunlop, James, and Thai Phung. "Phosphate and slow vacuolar channels in Beta vulgaris." Functional Plant Biology 25, no. 6 (1998): 709. http://dx.doi.org/10.1071/pp97046.
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Movement of phosphate through slow vacuolar (SV) ion channels and the effects of phosphate on SV currents were investigated using vacuoles from Beta vulgaris L. When the vacuoles contained 50 mM phosphate, the addition of phosphate to the bath shifted the apparent reversal potential for whole vacuole currents to more positive values, suggesting an outward rectifying current due to movement of phosphate ions out of the vacuole. However absolute values for reversal potentials obtained from the current-voltage curves and tail currents for whole vacuoles suggested that the membranes were relatively impermeable to phosphate. Single-channel data showed that the vacuole preparations contained more than one species of ion channel and therefore the whole-vacuole data will not give definitive information about individual species of ion channels. One of the channels had a single channel reversal potential that indicated a permeability of H2PO4- ions relative to Cl- of 7. The probability of this outwardly rectifying channel being open had a marked dependence on voltage and, in these experiments, it was effectively closed for potentials negative of +20 mV. The single-channel conductance was 19.4 ± 3.1 pS with 50 mM KH2PO4 in the vacuole and 10 mM total phosphate concentration in the bath. A channel with these characteristics has not been reported previously. In addition to the data identifying a phosphate channel, it was found that the presence of phosphate in the bath solution slowed the rate of activation of the SV currents. This effect was partially reversed when phosphate was removed from the bath.
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Palmer, L. G., H. Choe, and G. Frindt. "Is the secretory K channel in the rat CCT ROMK?" American Journal of Physiology-Renal Physiology 273, no. 3 (September 1, 1997): F404—F410. http://dx.doi.org/10.1152/ajprenal.1997.273.3.f404.
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The biophysical properties of low-conductance secretory K (SK) channels in the apical membrane of the rat cortical collecting tubule were examined to compare these properties with those of the cloned renal K channels of the ROMK family expressed in oocytes. At room temperature, with the tubule superfused with 140 mM K and 110 mM cation in the pipette, the inward single-channel conductance of the SK channels was 36 +/- 1 pS for K, 41 +/- 2 pS for NH4, and 22 +/- 3 pS for Tl. The reversal potential was nearly the same for K and Tl in the pipette but was shifted by -60 mV for NH4. The kinetics of the channel when K was the permeant ion could be described by a single open state (mean open time, 24 ms) and two closed states (mean closed times, 1.6 and 65 ms). The kinetics of SK changed when Tl was the permeant ion (mean open times of 6.6 ms and no long closed state) and when NH4 was the permeant ion (mean open time of 3.0 ms and a more prevalent long closed state). Thus the gating kinetics of the channel depend strongly on the nature of the conducted ion. The properties of SK channels were quite similar to those of ROMK2 expressed in Xenopus oocytes and measured under similar conditions, suggesting that these channels are identical.
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Chtanko, N., M. E. Toimil-Molares, T. W. Cornelius, D. Dobrev, and R. Neumann. "Ion-track based single-channel templates for single-nanowire contacting." Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 236, no. 1-4 (July 2005): 103–8. http://dx.doi.org/10.1016/j.nimb.2005.03.264.
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ABBOTT, GEOFFREY W., and STEVE A. N. GOLDSTEIN. "A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs)." Quarterly Reviews of Biophysics 31, no. 4 (November 1998): 357–98. http://dx.doi.org/10.1017/s0033583599003467.
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1. INTRODUCTION 3581.1 Summary 3581.2 Overview 3591.3 Four classes of pore-forming K+channel subunits – necessary and (sometimes) sufficient 3611.4 Soluble and peripheral membrane proteins that interact with P loop subunits to alter function 3621.5 Integral membrane proteins that interact with P loop subunits to alter function 3632. MinK DETERMINES THE FUNCTION OF MIXED CHANNEL COMPLEXES 3632.1 The KCNE1 gene product (MinK) gives rise to K+-selective currents and controversy 3632.2 MinK assembles with a P loop protein, KvLQT1, to form K+channels with unique function 3642.2.1 Single-channel conductance of KvLQT1 and MinK/KvLQT1 channels 3662.2.2 Other differences between KvLQT1 and MinK/KvLQT1 channels 3672.3 MinK assembles with HERG, another P loop subunit, to regulate channel activity 3682.4 MinK does not form chloride-selective ion channels 3683. EXPERIMENTAL AND NATURAL MinK MUTATIONS 3693.1 Site-directed mutations 3693.1.1 MinK mutation alters basic channel attributes and identifies key residues 3693.1.2 MinK is a Type I transmembrane peptide 3703.1.3 MinK is intimately associated with the IKspore 3703.1.4 The number of MinK subunits in IKschannel complexes 3723.2 KCNE1 mutations associated with arrhythmia and deafness alter IKschannel function 3733.3 Summary of MinK sites critical to IKschannel function 3744. MinK-RELATED PEPTIDES: AN EMERGING SUPERFAMILY 3744.1 KCNE2, 3 and 4 encode MinK-related peptides 1, 2 and 3 (MiRPs) 3744.2 MiRP1 assembles with a P loop protein, HERG, to form K+channels with unique function 3754.2.1 MiRP1 alters activation, deactivation and single-channel conductance 3764.2.2 MiRP1 alters regulation by K+ion and confers biphasic kinetics to channel blockade 3784.2.3 Stable association of MiRP1 and HERG subunits 3804.3 KCNE2 mutations are associated with arrhythmia and decreased K+flux 3834.4 Summary of the evidence that cardiac IKrchannels are MiRP1/HERG complexes 3855. MinK-RELATED PEPTIDES: COMMONALTIES AND IMPLICATIONS 3865.1 Genetics and structure 3865.2 Cell biology and function 3876. ANSWERS, SOME OUTSTANDING ISSUES, CONCLUSIONS 3877. ACKNOWLEDGEMENTS 3898. REFERENCES 389MinK and MinK-related peptide 1 (MiRP1) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRP1/HERG complexes reproduce the cardiac currents called IKs and IKr, respectively. Inherited mutations in KCNE1 (encoding MinK) and KCNE2 (encoding MiRP1) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily.
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Demuro, Angelo, and Ian Parker. "“Optical Patch-clamping”." Journal of General Physiology 126, no. 3 (August 15, 2005): 179–92. http://dx.doi.org/10.1085/jgp.200509331.
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We describe an optical technique using total internal reflection fluorescence (TIRF) microscopy to obtain simultaneous and independent recordings from numerous ion channels via imaging of single-channel Ca2+ flux. Muscle nicotinic acetylcholine (ACh) receptors made up of αβγδ subunits were expressed in Xenopus oocytes, and single channel Ca2+ fluorescence transients (SCCaFTs) were imaged using a fast (500 fps) electron-multiplied c.c.d. camera with fluo-4 as the indicator. Consistent with their arising through openings of individual nicotinic channels, SCCaFTs were seen only when a nicotinic agonist was present in the bathing solution, were blocked by curare, and increased in frequency as roughly the second power of [ACh]. Their fluorescence amplitudes varied linearly with membrane potential and extrapolated to zero at about +60 mV. The rise and fall times of fluorescence were as fast as 2 ms, providing a kinetic resolution adequate to characterize channel gating kinetics; which showed mean open times of 7.9 and 15.8 ms when activated, respectively, by ACh or suberyldicholine. Simultaneous records were obtained from >400 channels in the imaging field, and we devised a novel “channel chip” representation to depict the resultant large dataset as a single image. The positions of SCCaFTs remained fixed (<100 nm displacement) over tens of seconds, indicating that the nicotinic receptor/channels are anchored in the oocyte membrane; and the spatial distribution of channels appeared random without evidence of clustering. Our results extend single-channel TIRFM imaging to ligand-gated channels that display only partial permeability to Ca2+, and demonstrate an order-of-magnitude improvement in kinetic resolution. We believe that functional single-channel imaging opens a new approach to ion channel study, having particular advantages over patch-clamp recording in that it is massively parallel, and provides high-resolution spatial information that is inaccessible by electrophysiological techniques.
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Nechyporuk-Zloy, Volodymyr, Christian Stock, Hermann Schillers, Hans Oberleithner, and Albrecht Schwab. "Single plasma membrane K+ channel detection by using dual-color quantum dot labeling." American Journal of Physiology-Cell Physiology 291, no. 2 (August 2006): C266—C269. http://dx.doi.org/10.1152/ajpcell.00633.2005.
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K+ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K+ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K+ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K+ channel structure contrasts with the difficulties to visualize single K+ channel proteins in their physiological environment. We describe a method to identify single Ca2+-activated K+ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K+ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level.
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Smeazzetto, Serena, Francesco Tadini-Buoninsegni, Gerhard Thiel, and Maria Rosa Moncelli. "Selectivity of the phospholamban ion channel investigated by single channel measurements." Journal of Electroanalytical Chemistry 812 (March 2018): 244–48. http://dx.doi.org/10.1016/j.jelechem.2018.01.028.
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Keller, Bernhard U., Rainer Hedrich, Winchil L. C. Vaz, and Manuel Criado. "Single channel recordings of reconstituted ion channel proteins: an improved technique." Pfl�gers Archiv European Journal of Physiology 411, no. 1 (January 1988): 94–100. http://dx.doi.org/10.1007/bf00581652.
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Duszyk, Marek, Andrew S. French, and S. F. Paul Man. "Cystic fibrosis affects chloride and sodium channels in human airway epithelia." Canadian Journal of Physiology and Pharmacology 67, no. 10 (October 1, 1989): 1362–65. http://dx.doi.org/10.1139/y89-217.
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Abnormalities of epithelial function in cystic fibrosis (CF) have been linked to defects in cell membrane permeability to chloride or sodium ions. Recently, a class of chloride channels in airway epithelial cells have been reported to lack their usual sensitivity to phosphorylation via cAMP-dependent protein kinase, suggesting that CF could be due to a single genetic defect in these channels. We have examined single chloride and sodium channels in control and CF human nasal epithelia using the patch-clamp technique. The most common chloride channel was not the one previously associated with CF, but it was also abnormal in CF cells. In addition, the number of sodium channels was unusually high in CF. These findings suggest a wider disturbance of ion channel properties in CF than would be produced by a defect in a single type of channel.Key words: ion channels, cystic fibrosis, airway, epithelium.
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Turman, Daniel L., Jacob T. Nathanson, Randy B. Stockbridge, Timothy O. Street, and Christopher Miller. "Two-sided block of a dual-topology F− channel." Proceedings of the National Academy of Sciences 112, no. 18 (April 20, 2015): 5697–701. http://dx.doi.org/10.1073/pnas.1505301112.
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The Fluc family is a set of small membrane proteins forming F−-specific electrodiffusive ion channels that rescue microorganisms from F− toxicity during exposure to weakly acidic environments. The functional channel is built as a dual-topology homodimer with twofold symmetry parallel to the membrane plane. Fluc channels are blocked by nanomolar-affinity fibronectin-domain monobodies originally selected from phage-display libraries. The unusual symmetrical antiparallel dimeric architecture of Flucs demands that the two chemically equivalent monobody-binding epitopes reside on opposite ends of the channel, a double-sided blocking situation that has never before presented itself in ion channel biophysics. However, it is not known if both sites can be simultaneously occupied, and if so, whether monobodies bind independently or cooperatively to their transmembrane epitopes. Here, we use direct monobody-binding assays and single-channel recordings of a Fluc channel homolog to reveal a novel trimolecular blocking behavior that reveals a doubly occupied blocked state. Kinetic analysis of single-channel recordings made with monobody on both sides of the membrane shows substantial negative cooperativity between the two blocking sites.
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Yue, D. T., and E. Marban. "Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+." Journal of General Physiology 95, no. 5 (May 1, 1990): 911–39. http://dx.doi.org/10.1085/jgp.95.5.911.
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We investigated the mechanism whereby ions cross dihydropyridine-sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole-fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi-ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis-Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole-fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.
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Vanuytsel, Steven, Christopher L. Parperis, and Mark I. Wallace. "Combined Single-Molecule FRET and Single-Channel Recording to Link Ion Channel Conformation and Function." Biophysical Journal 118, no. 3 (February 2020): 466a—467a. http://dx.doi.org/10.1016/j.bpj.2019.11.2591.
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Clausen, Michael V., Viwan Jarerattanachat, Elisabeth P. Carpenter, Mark S. P. Sansom, and Stephen J. Tucker. "Asymmetric mechanosensitivity in a eukaryotic ion channel." Proceedings of the National Academy of Sciences 114, no. 40 (September 18, 2017): E8343—E8351. http://dx.doi.org/10.1073/pnas.1708990114.
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Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K+ channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli.
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Davis, Ryan W., Elizabeth L. Patrick, Lauren A. Meyer, Theodore P. Ortiz, Jason A. Marshall, David J. Keller, Susan M. Brozik, and James A. Brozik. "Thermodynamic Properties of Single Ion Channel Formation: Gramicidin." Journal of Physical Chemistry B 108, no. 39 (September 2004): 15364–69. http://dx.doi.org/10.1021/jp049686y.
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Schewe, Phil F. "The activity of a single ion-channel protein." Physics Today 56, no. 2 (February 2003): 9. http://dx.doi.org/10.1063/1.4796979.
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