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 large enough for reducing series resistance when recording large ion currents. Tubes with an inner diameter of 2-5 mm are usually acceptable.</li>
	</ol>
	</li>
	<li>Preparation of agarose 3 M KCl solution
	<ol>
		<li>Prepare 100 mL of 3 M KCl solution (1 M or 2 M is also acceptable).</li>
		<li>Weigh 3&#160;g of agarose.</li>
		<li>Dissolve the agarose in 100 mL of KCl (i.e.,&#160;3% agarose) on a hot plate between 90 and 100 &#176;C.</li>
	</ol>
	</li>
	<li>Loading the bridges with 3 M KCl agarose
	<ol>
		<li>For easy loading, immerse the U-shaped glass bridges in the agarose-KCl solution. 
		NOTE: It is easy to dig out the glass bridges if the agarose-KCl solution is contained in a shallow and broad container.</li>
		<li>Keep them overnight at room temperature (RT) for the agarose to set and harden.</li>
		<li>Carefully dig out the agarose-KCl-loaded glass bridges from the set/hardened agarose-salt.</li>
	</ol>
	</li>
	<li>Storing the bridges
	<ol>
		<li>Prepare enough volume (i.e., 500 mL) of the 3 M KCl solution in a wide-necked bottle.</li>
		<li>Store the prepared agarose-salt bridges in the bottle in a refrigerator.</li>
	</ol>
	</li>
</ol>
2. Application of Fluid Flow Shear Force to Cells in a Patch-Clamping Chamber
NOTE: A schematic diagram of the patch-clamp experimental set-up is shown in Figure 1.
<ol>
	<li>Place a container loaded with bathing solution (volume and height should already be measured) above the patch-clamp chamber.</li>
	<li>Fill the patch-clamp chamber with the bathing solution by suctioning the tube.</li>
	<li>To stop the fluid flow, clip the tube at the container&#8217;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 &#8220;stationary&#8221; control condition.</li>
	<li>To apply fluid flow shear force, open both tubes on the container and suction sides at the same time.</li>
	<li>Before or after applying the fluid flow shear force to the cell, measure the flow rate in mL/min.</li>
	<li>Calculate the flow rate by measuring the decrease in fluid volume over a given time.</li>
	<li>From the measured flow rate and geometry (structure) of the bathing chamber, the shear force applied to the cell by the fluid flow should be estimated (see discussion section).</li>
	<li>Alternatively, to control the flow rate (for steps 2.3-2.6), use a perfusion pump. In this case, be careful to ensure a constant rather than a pulsatile flow.</li>
</ol>
3. Measuring Changes in Liquid-Metal Junction Potential by Fluid Flow Between Bath Solution and Ag/AgCl Reference Electrode (Figure 3A)
<ol>
	<li>Use the Ag/AgCl electrode or pellet, which is available from the ready-made products, without the agarose salt bridge.</li>
	<li>Prepare a normal physiological salt saline for the bathing chamber (e.g., 143 mM NaCl, 5.4 mM KCl, 0.33 mM NaH2PO4, 5 mM HEPES, 0.5 mM MgCl2, 1.8 mM CaCl2, 11 mM D-glucose; pH adjusted to 7.4 with NaOH).</li>
	<li>Place a patch pipette containing a 3 M KCl solution in the chamber to minimize the junction potential shift between the pipette and bathing solutions.</li>
	<li>Fix the voltage-clamp amplifier to the current clamp mode (&#8220;I = 0&#8221; or &#8220;CC&#8221;).</li>
	<li>After nullifying the initial offset potential, measure changes in voltage induced by various flow rates.</li>
	<li>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 Ag/AgCl electrode.</li>
</ol>
4. Experimental Estimation of Real Cl- Concentration in the Unstirred Layer Adjacent to Ag/AgCl Electrode Under Static Condition (Figure 3B)
<ol>
	<li>From the results of step 3, draw the junction potential-flow rate relationships and estimate the maximal (saturating) value of junction potential shift by the supra-fluid flow rate.</li>
	<li>Prepare solutions with various concentrations of Cl (i.e., 50, 99, 147, 195, and 288 mM of NaCl).</li>
	<li>By changing the Cl- concentration in the bathing fluid, draw the junction potential-[Cl-] relationship. Note that the fluid rate should be constant and sufficiently high (&#62; 30 mL/min) to prevent the decrease of Cl- concentration to that of the adjacent Ag/AgCl reference electrode.</li>
	<li>From the two relationship curves, estimate the changes in Cl- concentration from the measured junction potential shift.</li>
</ol>

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	<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. L., O&#8217;Donnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductions+in+arterial+diameter+produced+by+chronic+decreases+in+blood+flow+are+endothelium-dependent.">Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent.</a> Science. 231, 405-407 (1986).</li><li>Pohl, U., 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=Crucial+role+of+endothelium+in+the+vasodilator+response+to+increased+flow+in+vivo.">Crucial role of endothelium in the vasodilator response to increased flow in vivo.</a> Hypertension. 8, 37-44 (1986).</li><li>Ranade, S. S., 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=a+mechanically+activated+ion+channel,+is+required+for+vascular+development+in+mice.">a mechanically activated ion channel, is required for vascular development in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 10347-10352 (2014).</li><li>Hoger, J. H., 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=Shear+stress+regulates+the+endothelial+Kir2.1+ion+channel.">Shear stress regulates the endothelial Kir2.1 ion channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (11), 7780-7785 (2002).</li><li>Mendoza, S. A., 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=TRPV4-mediated+endothelial+Ca2++influx+and+vasodilation+in+response+to+shear+stress.">TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress.</a> American Journal of Physiology-Heart and Circulatory Physiology. 298, H466-H476 (2010).</li><li>Brahler, S., 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=Genetic+deficit+of+SK3+and+IK1+channels+disrupts+the+endothelium-derived+hyperpolarizing+factor+vasodilator+pathway+and+causes+hypertension.">Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and causes hypertension.</a> Circulation. 119, 2323-2332 (2009).</li><li>Lee, S., 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+pressure+modulates+L-type+Ca2++channel+via+enhancement+of+Ca2+-induced+Ca2++release+in+rat+ventricular+myocytes.">Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes.</a> American Journal of Physiology-Cell Physiology. 294, C966-C976 (2008).</li><li>Kim, J. 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>

The authors have nothing to disclose.

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><tbody><tr><td>RC-11 open bath chamber</td><td>Warner instruments, USA</td><td>W4 64-0307</td><td></td></tr><tr><td>Ag/AgCl electrode pellet</td><td>World Precision Instruments, USA</td><td>EP1</td><td></td></tr><tr><td>Agarose</td><td>Sigma-aldrich, USA</td><td>A9793</td><td></td></tr><tr><td>Voltage-clamp amplifier</td><td>HEKA, Germany</td><td>EPC8</td><td></td></tr><tr><td>Voltage-clamp amplifier</td><td>Molecular Devices, USA</td><td>Axopatch 200B</td><td></td></tr><tr><td>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. ...

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Biochemistry

6.3K Views.  Konkuk University School of Medicine. Dit indienen kan helpen bij het beantwoorden van belangrijke vragen in de elektrofysiologische sfeer, zoals de vloeistofstroom in alleen de regelgeving van ionkanalen. Het belangrijkste voordeel van deze techniek is dat het de werkelijke ionenconcentratie in de ongeerdateerde grenslaag van het membraanoppervlak kan schatten voor gegevensinterpretatie met betrekking tot vloeistofstroomregulatie van ionenkanalen. Om deze procedure te beginnen, buigt u de vuurglas capillaire buis om een U-vorm t...

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Biology

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Neuroscience

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, electrophysiologically accessing these channels within small intracellular vesicles has been a challenge. The development of endolysosomal patch-clamp techniques has been instrumental in overcoming this barrier, allowing for the direct measurement of ion channel activity in endolysosomal membranes.
Compared to existing planar patch-clamp techniques, endolysosomal patch-clamp can simultaneously record multiple cells and easily combine with other measurement methods. Manual operation offers the advantage of visualizing targeted vesicles. It also addresses the limitation of the indispensable presence of Ca2+ on one side of the endolysosomal membrane, increasing the flexibility of experimental design. Utilizing endolysosomal patch-clamp techniques enables the direct measurement and analysis of ion channel activity within endolysosomes.
Given the close link between aberrant endolysosomal ion channel function and diseases such as neurodegenerative diseases and metabolic disorders, investigating and modulating these channels may unveil new drug targets. By restoring intracellular ion balance, we may alleviate or cure related diseases. Therefore, this technique is pivotal for discovering new drug targets and developing relevant medications.

This protocol details a method for directly measuring ion channel activity on intracellular vesicles using a manual endolysosomal patch-clamp system. We illustrate the method that involves enlarging endolysosomes and manually isolating these vesicles. This approach ensures that researchers can accurately replicate and apply the procedure.

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, ...

Patch Clamp Electrophysiology

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply suction to form a high resistance seal. This process isolates a small &#8220;patch&#8221; of membrane that contains one or more ion channels. Using an electrode within the micropipette, researchers can &#8220;clamp&#8221; or control the electrical properties of the membrane, which is important for analysis of channel activity. The electrode also allows for changes in the voltage across the membrane, or the flow of ions through the membrane, to be recorded. This video begins with an overview of the principles behind patch clamp electrophysiology, an introduction to the necessary equipment, and descriptions of the various patch configurations, including whole cell, cell-attached, perforated, inside-out, and outside-out patches. Next, the key steps of a typical whole-cell patch clamp experiment are outlined, in which a current-voltage (IV) curve is generated. Finally, applications of patch clamp recording are provided to demonstrate how the biophysical properties of ion channels, cell excitability, and neuroactive compounds are evaluated in neurophysiology labs today.

Patch Clamp Electrophysiology: Principle &amp; Applications

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One ...

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

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