| 1. |
- Jansson, Erik, 1984, et al.
(författare)
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Effect of cholesterol depletion on the pore dilation of TRPV1
- 2013
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Ingår i: Molecular Pain. - : SAGE Publications. - 1744-8069. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
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| 2. |
- Bridle, Helen, 1979, et al.
(författare)
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On-chip fabrication to add temperature control to a microfluidic solution exchange system
- 2008
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Ingår i: Lab on a Chip - Miniaturisation for Chemistry and Biology. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 8:3, s. 480-483
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Tidskriftsartikel (refereegranskat)abstract
- We present a concept for the post production modification of commercially available microfluidic devices to incorporate local temperature control, thus allowing for the exact alignment of heating structures with the existing features, e.g. wells, channels or valves, of a system. Specifically, we demonstrate the application of programmable local heating, controlled by computerized PI regulation, to a rapid solution exchanger. Characterisation of the system to show that both uniform temperature distributions and temperature gradients can be established, and to confirm that the solution exchange properties are undisturbed by heating, was achieved using in situ thermometry and amperometry. © The Royal Society of Chemistry.
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| 3. |
- Granfeldt, D., et al.
(författare)
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Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp Protocols
- 2006
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:23, s. 7947-7953
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Tidskriftsartikel (refereegranskat)abstract
- Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABA(A) receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABA(A) receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABA(A) receptors.
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| 4. |
- Jansson, Erik, 1984, et al.
(författare)
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Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes
- 2012
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:13, s. 5582-5588
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50–150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm2, and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1–1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect 65% more unique membrane-associated protein (p
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| 5. |
- Millingen, Maria, 1980, et al.
(författare)
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Ligand-specific temperature-dependent shifts in EC50 values for the GABA(A) receptor
- 2008
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 80:1, s. 340-343
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Tidskriftsartikel (refereegranskat)abstract
- We introduce a system for temperature control of a commercial microfluidic superfusion device that, in combination with patch-clamp, enables rapid acquisition of dose-response data at different temperatures. We obtained dose-response curves for the GABA A receptor, a ligand-gated ion channel, for two different agonists at temperatures between 25 and 40°C. For GABA, the dose-response curves shifted toward higher EC 50 values as the temperature increased, whereas for/?-alanine, the EC 50 values were constant. This shows that temperature is an important factor for obtaining accurate estimations of EC 50 values and also that such temperature effects can be ligand-specific. Using the EC 50 values, we estimated the enthalpy of dissociation between the ligand and the receptor. Furthermore, the technology introduced here is generally applicable to all patch-clamp studies where temperature control is desirable, e.g., studies of kinetics and thermodynamics, drug screening, compliant ADME/Tox testing, and in studies of temperature-gated ion channels. © 2008 American Chemical Society.
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| 6. |
- Millingen, Maria, 1980
(författare)
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Patch-clamp studies of the GABAA receptor using microfluidic methods
- 2007
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ion channels are membrane proteins that passively transport ions. When activated, conformational changes within the protein lead to opening of a channel pore, and the flow of ions causes a change in the membrane potential. This is the fundamental process behind the generation and transduction of nerve impulses. The GABAA receptor is a ligand-gated ion channel which is activated by the inhibitory neurotransmitter γ-amino-n-butyric acid (GABA). This receptor is mainly situated in the post-synaptic membrane and conducts inhibitory synaptic currents. Understanding the kinetics of ion channels like the GABAA receptor is important in order to understand what output, in terms of the shape and length of the synaptic current, will be the result of different inputs, in terms of the concentration profile of the neurotransmitter in the synaptic cleft. The GABAA receptor exhibits binding, gating, and desensitization, i.e. transit into long-lived, non-conducting, states. Desensitization has been suggested to regulate the length and amplitude of the synaptic GABAA currents, and also to reduce GABAA currents during repetitive GABA application.Using a combination of the patch-clamp technique and a microfluidic superfusion system, we have performed scanning experiments across gradients of the GABAA receptor agonists GABA and β-alanine. Results show that the order of application of different ligand concentrations, as well as the time and frequency of stimulations, affect the appearance of the dose-response curves and hence the EC50 values obtained by fitting data to the Hill equation. Also, repetitive applications cause slow desensitization which constitutes a memory function which lasts for minutes. By adding temperature control to the microfluidic chip, we have also obtained dose-response curves for temperatures between 25°C and 40°C. The EC50 value proved to be temperature dependent for GABA, but not for β-alanine. This could be due to differences in binding kinetics for GABA and β-alanine.
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| 7. |
- Millingen, Maria, 1980
(författare)
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Studies of ligand- and temperature-gated ion channels using microfluidic methods
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the studies of the GABAA receptor, a ligand-gated ion channel, and the ligand- and temperature-gated ion channel TRPV1. The desensitization behavior of both of these proteins has been studied for different conditions, using a microfluidic device for superfusion. Also, temperature control has been added to this system in order to enable studies of thermodynamic and kinetic properties of ion channels, as well as for studies of temperature-gated channels.We examine the effect of desensitization on dose-response curves for the GABAA receptor, and also compares two different GABAA receptor agonists, GABA and β-alanine, which display different affinities but similar efficacies and desensitization behavior. The results show that the dose-response curves depend greatly on the order of application, and computer simulations using a proposed kinetic model show that this is due to different distributions of the slow and fast desensitized states. This leads to a memory effect for the receptor population that can last up to minutes. We also describe the differential blocking effect of the competitive antagonist bicuculline on the different GABAA receptor state distributions and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. A concept for the post-production modification of commercially available microfluidic devices to incorporate local temperature control is presented and the resulting system characterized. This device was used to demonstrate that temperature affects the dose-response curves for the GABAA receptor in combination with GABA, but not with β-alanine, and this could be due to their different activation energies for binding and dissociation. The device was also used to characterize desensitization of the temperature-gated ion channel TRPV1 in response to low pH. Our results indicate that temperature affects the rate of both TRPV1 activation and desensitization, and that tachyphylaxis is both pH and temperature dependent. Also, if TRPV1 is exposed several times to low pH, the desensitization becomes slower, whereas the activation rate is constant.
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| 8. |
- Sinclair, J., et al.
(författare)
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A biohybrid dynamic random access memory
- 2006
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 128:15, s. 5109-5113
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Tidskriftsartikel (refereegranskat)abstract
- We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.
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