Ion Channel Sensor on a Silicon Support

2004 ◽  
Vol 820 ◽  
Author(s):  
Michael Goryll ◽  
Seth Wilk ◽  
Gerard M. Laws ◽  
Stephen M. Goodnick ◽  
Trevor J. Thornton ◽  
...  
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Chemical Vapor ◽  
Fast Method ◽  
Fluorocarbon Films ◽  
Novel Approach ◽  
Voltage Dependent ◽  
Assembled Monolayers

AbstractWe are building a biosensor based on ion channels inserted into lipid bilayers that are suspended across an aperture in silicon. The process flow only involves conventional optical lithography and deep Si reactive ion etching to create micromachined apertures in a silicon wafer. In order to provide surface properties for lipid bilayer attachment that are similar to those of the fluorocarbon films that are currently used, we coated the silicon surface with a fluoropolymer using plasma-assisted chemical vapor deposition. When compared with the surface treatment methods using self-assembled monolayers of fluorocarbon chemicals, this novel approach towards modifying the wettability of a silicon dioxide surface provides an easy and fast method for subsequent lipid bilayer formation. Current-Voltage measurements on OmpF ion channels incorporated into these membranes show the voltage dependent gating action expected from a working porin ion channel.

scholarly journals Proper Voltage-Dependent Ion Channel Function in Dysferlin-Deficient Cardiomyocytes

2015 ◽  
Vol 36 (3) ◽  
pp. 1049-1058 ◽  
Author(s):  
Lena Rubi ◽  
Vaibhavkumar S. Gawali ◽  
Helmut Kubista ◽  
Hannes Todt ◽  
Karlheinz Hilber ◽  
...  
Keyword(s):  
Ion Channels ◽  
Muscular Dystrophy ◽  
Ion Channel ◽  
Cardiac Electrophysiology ◽  
Cardiac Complications ◽  
Membrane Repair ◽  
Patch Clamp Technique ◽  
Cardiac Ion Channels ◽  
Ventricular Cardiomyocytes ◽  
Voltage Dependent

Background/Aims: Dysferlin plays a decisive role in calcium-dependent membrane repair in myocytes. Mutations in the encoding DYSF gene cause a number of myopathies, e.g. limb-girdle muscular dystrophy type 2B (LGMD2B). Besides skeletal muscle degenerative processes, dysferlin deficiency is also associated with cardiac complications. Thus, both LGMD2B patients and dysferlin-deficient mice develop a dilated cardiomyopathy. We and others have recently reported that dystrophin-deficient ventricular cardiomyocytes from mouse models of Duchenne muscular dystrophy show significant abnormalities in voltage-dependent ion channels, which may contribute to the pathophysiology in dystrophic cardiomyopathy. The aim of the present study was to investigate if dysferlin, like dystrophin, is a regulator of cardiac ion channels. Methods and Results: By using the whole cell patch-clamp technique, we compared the properties of voltage-dependent calcium and sodium channels, as well as action potentials in ventricular cardiomyocytes isolated from the hearts of normal and dysferlin-deficient (dysf) mice. In contrast to dystrophin deficiency, the lack of dysferlin did not impair the ion channel properties and left action potential parameters unaltered. In connection with normal ECGs in dysf mice these results suggest that dysferlin deficiency does not perturb cardiac electrophysiology. Conclusion: Our study demonstrates that dysferlin does not regulate cardiac voltage-dependent ion channels, and implies that abnormalities in cardiac ion channels are not a universal characteristic of all muscular dystrophy types.

scholarly journals Development of an automated system to measure ion channel currents using a surface-modified gold probe

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Minako Hirano ◽  
Masahisa Tomita ◽  
Chikako Takahashi ◽  
Nobuyuki Kawashima ◽  
Toru Ide
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayer ◽  
Measurement System ◽  
Automated System ◽  
Channel Activity ◽  
Channel Current ◽  
Gold Probe ◽  
Measurement Efficiency ◽  
Channel Currents

AbstractArtificial lipid bilayer single-channel recording technique has been employed to determine the biophysical and pharmacological properties of various ion channels. However, its measurement efficiency is very low, as it requires two time-consuming processes: preparation of lipid bilayer membranes and incorporation of ion channels into the membranes. In order to address these problems, we previously developed a technique based on hydrophilically modified gold probes on which are immobilized ion channels that can be promptly incorporated into the bilayer membrane at the same time as the membrane is formed on the probes’ hydrophilic area. Here, we improved further this technique by optimizing the gold probe and developed an automated channel current measurement system. We found that use of probes with rounded tips enhanced the efficiency of channel current measurements, and introducing a hydrophobic area on the probe surface, beside the hydrophilic one, further increased measurement efficiency by boosting membrane stability. Moreover, we developed an automated measurement system using the optimized probes; it enabled us to automatically measure channel currents and analyze the effects of a blocker on channel activity. Our study will contribute to the development of high-throughput devices to identify drug candidates affecting ion channel activity.

Ion-channel properties of mastoparan, a 14-residue peptide from wasp venom, and of MP3, a 12-residue analogue

1990 ◽  
Vol 239 (1296) ◽  
pp. 383-400 ◽  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Type I ◽  
Type Ii ◽  
Channel Formation ◽  
Channel Inactivation ◽  
Ion Channel Properties ◽  
Discrete Channel ◽  
Voltage Dependent ◽  
Channel Properties

Mastoparan, a 14-residue peptide, has been investigated with respect to its ability to form ion channels in planar lipid bilayers. In the presence of 0.3 - 3.0 μ M mastoparan, two types of activity are seen. Type I activity is characterized by discrete channel openings, exhibiting multiple con­ductance levels in the range 15-700 pS. Type II activity is characterized by transient increases in bilayer conductance, up to a maximum of about 650 pS. Both type I and type II activities are voltage dependent. Channel activation occurs if the compartment containing mastoparan is held at a positive potential; channel inactivation if the same compartment is held at a negative potential. Channel formation is dependent on ionic strength; channel openings are only observed at KCl concentrations of 0.3 M or above. Furthermore, raising the concentration of KCl to 3.0 M stabilizes the open form of the channel. Mastoparan channels are weakly cation selective, P K/Cl ≈ 2. A 12-residue analogue, des -Ile 1 , Asn 2 mastoparan, preferentially forms type I channels. The ion channels formed by these short peptides may be modelled in terms of bundles of transmembrane α -helices.

Distribution and modulation of ion channel and receptor topography on nerve cell surfaces

1993 ◽  
Vol 51 ◽  
pp. 34-35
Author(s):  
Kimon J. Angelides ◽  
Barry Hicks
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Dendritic Spines ◽  
Neuronal Membrane ◽  
Synaptic Membrane ◽  
Nodes Of Ranvier ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels ◽  
Synaptic Junctions ◽  
Homogeneous Distributions

The distribution of ion channels and receptors over the neuronal surface is important for the receipt of incoming synaptic inputs and for the integration of these inputs. Most voltage-gated and ligand-gated ion channels have non-homogeneous distributions in the neuronal membrane, many being restricted to either dendritic, axonal or somatic domains and further localized within these domains to regions such as dendritic spines, nodes of Ranvier or synaptic junctions (1-3). For example voltage-dependent calcium channels are localized and immobilized on dendrites (4), while voltage-dependent sodium channel are localized on axon hillocks (5) and nodes of Ranvier. Determining where and how ion channels are distributed and maintained is important for a variety of reasons. Ion channels in growth cones have a role in neurite outgrowth mechanisms (6), they are obligatory for synaptic transmission and they are required for amplification of neurotransmitter signals in the post synaptic membrane (7). Changes in ion channel distributions are an important aspect in development and plasticity (8,9).

Ion channels ofFasciola hepaticaincorporated into planar lipid bilayers

Parasitology ◽  
2004 ◽  
Vol 128 (1) ◽  
pp. 83-89 ◽  
Author(s):  
J. H. JANG ◽  
S. D. KIM ◽  
J. B. PARK ◽  
S. J. HONG ◽  
P. D. RYU
Keyword(s):  
Ion Channels ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Fasciola Hepatica ◽  
State Probability ◽  
Planar Lipid Bilayer ◽  
Single Channels ◽  
Permeable Channel ◽  
Important Target ◽  
Large Conductance Channel

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.

scholarly journals Thiazolidinedione insulin sensitizers alter lipid bilayer properties and voltage-dependent sodium channel function: implications for drug discovery

2011 ◽  
Vol 138 (2) ◽  
pp. 249-270 ◽  
Author(s):  
Radda Rusinova ◽  
Karl F. Herold ◽  
R. Lea Sanford ◽  
Denise V. Greathouse ◽  
Hugh C. Hemmings ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Gene Activation ◽  
Transport Processes ◽  
Titration Calorimetry ◽  
Peroxisome Proliferator ◽  
Inactivation Curve ◽  
Channel Function ◽  
Voltage Dependent ◽  
Target Effects

The thiazolidinediones (TZDs) are used in the treatment of diabetes mellitus type 2. Their canonical effects are mediated by activation of the peroxisome proliferator–activated receptor γ (PPARγ) transcription factor. In addition to effects mediated by gene activation, the TZDs cause acute, transcription-independent changes in various membrane transport processes, including glucose transport, and they alter the function of a diverse group of membrane proteins, including ion channels. The basis for these off-target effects is unknown, but the TZDs are hydrophobic/amphiphilic and adsorb to the bilayer–water interface, which will alter bilayer properties, meaning that the TZDs may alter membrane protein function by bilayer-mediated mechanisms. We therefore explored whether the TZDs alter lipid bilayer properties sufficiently to be sensed by bilayer-spanning proteins, using gramicidin A (gA) channels as probes. The TZDs altered bilayer elastic properties with potencies that did not correlate with their affinity for PPARγ. At concentrations where they altered gA channel function, they also altered the function of voltage-dependent sodium channels, producing a prepulse-dependent current inhibition and hyperpolarizing shift in the steady-state inactivation curve. The shifts in the inactivation curve produced by the TZDs and other amphiphiles can be superimposed by plotting them as a function of the changes in gA channel lifetimes. The TZDs’ partition coefficients into lipid bilayers were measured using isothermal titration calorimetry. The most potent bilayer modifier, troglitazone, alters bilayer properties at clinically relevant free concentrations; the least potent bilayer modifiers, pioglitazone and rosiglitazone, do not. Unlike other TZDs tested, ciglitazone behaves like a hydrophobic anion and alters the gA monomer–dimer equilibrium by more than one mechanism. Our results provide a possible mechanism for some off-target effects of an important group of drugs, and underscore the importance of exploring bilayer effects of candidate drugs early in drug development.

scholarly journals Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines

2016 ◽  
Vol 116 (08) ◽  
pp. 272-284 ◽  
Author(s):  
Joy R. Wright ◽  
Stefan Amisten ◽  
Alison H. Goodall ◽  
Martyn P. Mahaut-Smith
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Cell Lines ◽  
Cell Types ◽  
Human Platelets ◽  
Mrna Levels ◽  
Low Copy Number ◽  
Voltage Dependent ◽  
Hel Cells ◽  
Platelet Aggregates

SummaryIon channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288–11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These “channelome” datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.Supplementary Material to this article is available online at www.thrombosis-online.com.

scholarly journals Estimating the voltage-dependent free energy change of ion channels using the median voltage for activation

2011 ◽  
Vol 139 (1) ◽  
pp. 3-17 ◽  
Author(s):  
Sandipan Chowdhury ◽  
Baron Chanda
Keyword(s):  
Free Energy ◽  
Ion Channels ◽  
Ion Channel ◽  
Free Energy Change ◽  
Energy Change ◽  
Displacement Curve ◽  
Charge Movement ◽  
Voltage Gated ◽  
State Process ◽  
Voltage Dependent

Voltage-gated ion channels are crucial for electrical activity and chemical signaling in a variety of cell types. Structure-activity studies involving electrophysiological characterization of mutants are widely used and allow us to quickly realize the energetic effects of a mutation by measuring macroscopic currents and fitting the observed voltage dependence of conductance to a Boltzmann equation. However, such an approach is somewhat limiting, principally because of the inherent assumption that the channel activation is a two-state process. In this analysis, we show that the area delineated by the gating charge displacement curve and its ordinate axis is related to the free energy of activation of a voltage-gated ion channel. We derive a parameter, the median voltage of charge transfer (Vm), which is proportional to this area, and prove that the chemical component of free energy change of a system can be obtained from the knowledge of Vm and the maximum number of charges transferred. Our method is not constrained by the number or connectivity of intermediate states and is applicable to instances in which the observed responses show a multiphasic behavior. We consider various models of ion channel gating with voltage-dependent steps, latent charge movement, inactivation, etc. and discuss the applicability of this approach in each case. Notably, our method estimates a net free energy change of approximately −14 kcal/mol associated with the full-scale activation of the Shaker potassium channel, in contrast to −2 to −3 kcal/mol estimated from a single Boltzmann fit. Our estimate of the net free energy change in the system is consistent with those derived from detailed kinetic models (Zagotta et al. 1994. J. Gen. Physiol. doi:10.1085/jgp.103.2.321). The median voltage method can reliably quantify the magnitude of free energy change associated with activation of a voltage-dependent system from macroscopic equilibrium measurements. This will be particularly useful in scanning mutagenesis experiments.

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