Name: measurement of ion concentration in the unstirred boundary layer with open patch-clamp pipette: implications in control of ion channels by fluid flow
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow at JoVE.

This research was supported by the Pioneer Research Center Program (2011-0027921), by Basic Science Research Programs (2015R1C1A1A02036887 and NRF-2016R1A2B4014795) through the National Research Foundation of Korea funded by the Ministry of Science, ICT &#38; Future Planning, and by a grant of the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (HI15C1540).

All experiments were performed in accordance with the institutional guidelines of Konkuk University.
1. Agarose Salt Bridges Between the Bath Solution and Ag/AgCl Reference Electrode
NOTE: Agarose 3M KCl salt bridges are produced as previously described12 with minor variations.
<ol>
	<li>Formation of bridges
	<ol>
		<li>Bend the fire glass capillary tubes to form a U-shape as appropriate. The inner diameter of the capillaries should be larg...

<ol>
	<li>Gerhold, K. A., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+Channels+in+Endothelial+Responses+to+Fluid+Shear+Stress.">Ion Channels in Endothelial Responses to Fluid Shear Stress.</a> Physiology (Bethesda). 31 (5), 359-369 (2016).</li><li>Garcia-Roldan, J. L., Bevan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+constriction+and+dilation+of+cerebral+resistance+arteries.">Flow-induced constriction and dilation of cerebral resistance arteries.</a> Circulation Research. 66, 1445-1448 (1990).</li><li>Langille, B. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+flow+facilitates+inward+rectifier+K++current+by+convectively+restoring+[K+]+at+the+cell+membrane+surface.">Fluid flow facilitates inward rectifier K+ current by convectively restoring [K+] at the cell membrane surface.</a> Scientific Report. 6, 39585 (2016).</li><li>Park, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+fluid+flow+on+voltage-dependent+calcium+channels+in+rat+vascular+myocytes:+fluid+flow+as+a+shear+stress+and+a+source+of+artifacts+during+patch-clamp+studies.">Effects of fluid flow on voltage-dependent calcium channels in rat vascular myocytes: fluid flow as a shear stress and a source of artifacts during patch-clamp studies.</a> Biochemical and Biophysical Research Communications. 358 (4), 1021-1027 (2007).</li><li>Barry, P. H., Hope, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroosmosis+in+membranes:+effects+of+unstirred+layers+and+transport+numbers.+I.+Theory.">Electroosmosis in membranes: effects of unstirred layers and transport numbers. I. Theory.</a> Biophysical Journal. 9 (5), 700-728 (1969).</li><li>Barry, P. H., Hope, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroosmosis+in+membranes:+effects+of+unstirred+layers+and+transport+numbers.+II.+Experimental.">Electroosmosis in membranes: effects of unstirred layers and transport numbers. II. Experimental.</a> Biophysical Journal. 9 (5), 729-757 (1969).</li><li>Barry, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+unstirred-layer+transport+number+equations+from+the+Nernst-Planck+flux+equations.">Derivation of unstirred-layer transport number equations from the Nernst-Planck flux equations.</a> Biophysical Journal. 74 (6), 2903-2905 (1998).</li><li>Barry, P. H., Diamond, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unstirred+layers+on+membrane+phenomena.">Effects of unstirred layers on membrane phenomena.</a> Physiological Reviews. 64 (3), 763-872 (1984).</li><li>Park, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caveolar+remodeling+is+a+critical+mechanotransduction+mechanism+of+the+stretch-induced+L-type+Ca2++channel+activation+in+vascular+myocytes.">Caveolar remodeling is a critical mechanotransduction mechanism of the stretch-induced L-type Ca2+ channel activation in vascular myocytes.</a> Pfl&#252;gers Archiv - European Journal of Physiology. 469 (5-6), 829-842 (2017).</li><li>. A procedure for the formation of agar salt bridges Available from: <a target="_blank" href="https://www.warneronline.com/pdf/whitepapers/agar_bridges.pdf">https://www.warneronline.com/pdf/whitepapers/agar_bridges.pdf</a> (2018)</li><li>Cunningham, K. S., Gotlieb, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+shear+stress+in+the+pathogenesis+of+atherosclerosis.">The role of shear stress in the pathogenesis of atherosclerosis.</a> Laboratory Investigation. 85 (1), 9-23 (2005).</li><li>Resnick, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shear+stress+and+the+vascular+endothelium:+for+better+and+for+worse.">Fluid shear stress and the vascular endothelium: for better and for worse.</a> Progress in Biophysics &amp; Molecular Biology. 81 (3), 177-199 (2003).</li></ol>

In this study, we demonstrated a method to measure real Cl- concentration in the unstirred layer adjacent to the Ag/AgCl reference electrode by determining the liquid-metal junction potential with an open patch-clamp pipette filled with a high KCl concentration. The change in Cl- concentration in the boundary layer can result in a shift of junction potential when switching from static to fluid-flow conditions. Simply using an agarose KCl bridge between the reference electrode and bathing fluid can p...

Fluid flow is an important environmental cue that controls many physiological and pathological processes such as fluid flow-induced vasodilation and fluid shear force-dependent vascular remodeling and development1,2,3,4,5. Although the molecular mechanisms for the biological responses to fluid flow shear force are not fully understood, it is believed that fluid flow-mediated regulation of ion channel gating may critically contribute to fluid flow-induced responses5,6,7,8. For example, activation of the endothelial inward rectifier Kir2.1 and Ca2+-activated K+ (KCa2.3, KCNN3) channels after Ca2+ influx by fluid flow has been suggested to contribute to fluid flow-induced vasodilation6,7,8. Therefore, many ion channels, especially mechanically-activated or -inhibited channels, have been studied in terms of fluid flow/shear force sensitivity with the patch-clamp technique6,9,10,11. However, depending on the experimental protocol performed during patch-clamp recording, outcomes and interpretation of the data on fluid flow-regulations of ion channels can be erroneous10,11.
One source of fluid flow-induced artifacts in patch-clamp recording is from the junction potential between the bath fluid and Ag/AgCl reference electrode11. It is generally believed that the liquid/metal junction potential between the bathing fluid and Ag/AgCl electrode is constant as the Cl- concentration of the bathing fluid is kept constant, considering the chemical response between the bathing solution and Ag/AgCl electrode to be:
Ag + Cl-&#8596; AgCl + electron (e-)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;(Equation 1)
However, in a case where the overall electrochemical reaction between the bathing solution and Ag/AgCl reference electrode (Equation 1) proceeds from left to right, the Cl- concentration of the bathing fluid adjacent to the Ag/AgCl reference electrode (unstirred boundary layer12,13,14,15) may be much lower than that in the bulk of bathing solution, unless enough convectional transport is ensured. Using an old or non-ideal Ag/AgCl electrode with inadequate chlorination of Ag may increase such a risk. This fluid flow-related artifact at the reference electrode, in fact, can be excluded by simply placing a conventional agarose-salt bridge between the bathing fluid and reference electrode, since the artifact is based on alterations in real Cl- concentration adjacent to the Ag/AgCl electrode11. The protocol presented in this study describes how to prevent the flow-related junction potential changes and measure real ion concentrations in the unstirred boundary layer.
After placing an agarose KCl bridge between the bathing fluid and Ag/AgCl reference electrode, there is another crucial factor that should be considered: just as the reference Ag/AgCl electrode acts like a Cl- electrode, the ion channels also can function like an ion-selective electrode. The situation of an unstirred boundary layer between the bathing fluid and Ag/AgCl reference electrode arises during the movement of ions between the extracellular and intracellular solutions through the membrane ion channels. This implies that caution should be used when interpreting the regulation of ion channels by fluid flow. As discussed in our previous study11, the movement of ions through a solution in which an electrochemical gradient is present can occur via three distinct mechanisms: diffusion, migration, and convection, where diffusion is the movement induced by concentration gradient, migration is the movement driven by electrical gradient, and convection is the movement through fluid-flow. Among these three transport mechanisms, convection mode contributes most to the movement of ions11 (&#62; 1,000 times greater than diffusion or migration under usual patch-clamp settings). This forms the theoretical basis of why junction potential between the bathing fluid and Ag/AgCl reference electrode can very under different static and fluid-flow conditions11.
As per the hypothesis proposed above, some facilitatory effects of fluid flow on the ion channel current may be inferred from the convective restoration of real ion concentrations adjacent to the channel inlet at the membrane surface (unstirred boundary layer)10. In this case, the fluid flow-induced effects on ion channel currents have simply arisen from electrochemical events, not from the regulation of ion channel gating. A similar idea was previously suggested by Barry and colleagues12,13,14,15 based on rigorous theoretical considerations and experimental evidence, also known as the unstirred layer or transport number effect. If some ion channels have sufficient single channel conductance and long enough open-time to provide sufficient transport rates through the channels (a faster transport rate in the membrane than in the unstirred membrane surface), a boundary layer effect may arise. Thus, the convection-dependent transport can contribute to the eventual fluid-flow-induced facilitations of ion current10,12,13,14,15.
In this study, we emphasize the importance of using an agar or agarose salt-bridge while studying fluid-flow-induced regulation of ion currents. We also provide a method for measuring real ion concentrations in the unstirred boundary layer adjacent to the Ag/AgCl reference electrode and membrane ion channels. Furthermore, the theoretical interpretation of fluid flow-induced modulation of ion channel currents (i.e., convection hypothesis or unstirred layer transport number effect) can provide valuable insights for designing and interpreting studies on the shear force-regulation of ion channels. According to the unstirred boundary layer transport number effect, we predict that ion channel currents through all types of membrane ion channels can be facilitated by fluid flow, independent of their biological sensitivity to fluid flow shear force, but only if the ion channels have sufficient single channel conductance and long open-time. Higher ion channel current densities may increase the unstirred boundary layer effect at the cell membrane surface.

<table border="0" cellpadding="0" cellspacing="0" style="width:784px;" width="783">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">RC-11 open bath chamber&#160;</td>
			<td style="width:229px;">Warner instruments, USA</td>
			<td style="width:149px;">W4 64-0307</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ag/AgCl electrode pellet</td>
			<td>World Precision Instruments, USA</td>
			<td>EP1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Agarose&#160;</td>
			<td>Sigma-aldrich, USA</td>
			<td>A9793</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Voltage-clamp amplifier&#160;</td>
			<td>HEKA, Germany</td>
			<td>EPC8</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">&#160;Voltage-clamp amplifier&#160;</td>
			<td>Molecular Devices, USA</td>
			<td>Axopatch 200B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Liquid pump</td>
			<td>KNF Flodos, Switzerland</td>
			<td>FEM08</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of ion concentration in the unstirred boundary layer with open patch-clamp pipette: implications in control of ion channels by fluid flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Whole cell voltage-dependent L-type Ca2+ channel (VDCCL) currents were recorded in the enzymatically dispersed rat mesenteric arterial myocytes, as previously described11. The arterial myocytes were dialyzed with a Cs-rich pipette solution under the nystatin-perforated configuration with divalent cation-free bathing solution to facilitate the current flow through VDCCL11,16....

Watch this Scientific Journal Video about Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow at JoVE.

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

This submittal can help answer key questions in the electro-physiologic sphere, such as the fluid flow in just the regulations of ion channels. The main advantage of this technique is that it can estimate the actual ion concentration in the unstirred boundary layer of the membrane surface for data interpretation related to fluid flow regulation of ion channels. To begin this procedure, bend the fired-glass capillary tube to form a U shape.The inner diameter of the capillary should be large enough for reducing series resistance when recording large ion currents. Next, dissolve three grams of agarose in 100 milliliters of three-molar potassium chloride, and place it on a hot plate between 90 and 100 degrees Celsius. Then, load the bridge with the potassium chloride agarose, by immersing the glass bridge in the solution.Keep it overnight at room temperature for the agarose to set and harden. The next day, carefully dig out the agarose potassium chloride-loaded glass bridge from the hardened agarose salt. Store the bridge in a wide-necked bottle of three-molar potassium chloride in the refrigerator.In this procedure, place a container loaded with bathing solution, above the patch-clamp chamber. Next, fill the patch-clamp chamber with the bathing solution by suctioning the tube. To stop the fluid flow, clip the tube at the container's side, to block the fluid flow.Then clip the tube at the suction side to stop the suction at the same time. This is the stationary control condition. To apply fluid flow shear force, open both tubes on the container and suction sides at the same time.Before or after applying the fluid flow shear force to the cell, calculate the flow rate in milliliter per minute by measuring the decrease in fluid volume over a given time. To measure changes in liquid-metal junction potential, prepare a normal physiological-salt saline for the bathing chamber, and compare them in the absence and presence of agar potassium chloride bridge. Next, place a patch pipette containing three-molar potassium chloride solution in the chamber, to minimize the junction potential shift between the pipette and bathing solutions.Then set the voltage clamp amplifier to the current clamp mode. After nullifying the initial offset potential, measure the changes in voltage induced by varying the flow rates. To verify that the changes in voltage are liquid-metal junction potentials, re-examine the effect of fluid flow on the junction potential, using the agarose salt bridge between the bath solution and the reference electrode.With the results of the changes in liquid-metal junction potential, draw the function potential flow rate relationships, and estimate the saturating value of junction potential shift by the supra-fluid flow rate. Then, change the chloride concentration in the bathing fluid, and draw the junction potential chloride concentration relationship. Note that the fluid rate should be constant, and sufficiently high to prevent the decrease of chloride concentration to that of the adjacent silver-silver chloride reference electrode.From the two relationship curves, estimate the changes in chloride concentration from the measured junction potential shift. VDCC-L currents were recorded in the enzymatically dispersed rat mesenteric arterial myocytes, with nystatin perforated patch-clamp recording. With agarose potassium chloride bridge, the junction potential between the reference electrode and bathing solutions could be minimized, and the fluid flow increased the VDCC-L current's voltage independently.However, when the silver-silver chloride reference electrode was directly connected to the bathing fluid, without an agarose potassium chloride bridge, the IV relationship in the presence of fluid flow shifted to the right, compared to that of the VDCC-L currents under a static condition. The fluid flow-induced increase of Kerr 2.1 currents, which were recorded in rat basophilic leukemia cells with the agarose bridge, can be explained by the unstirred layer effect. After its development, this technique paved the way for researchers in the over electro-physiology.So it's for the regulation of fluid flow in the ion channel currents. In terms of electro-chemical phenomena, in the unstirred boundary layer at cell membrane surface.

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Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

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Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

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The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

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The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...