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>
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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>

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 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....

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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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6.2K 조회수.  Konkuk University School of Medicine. 이 제출은 이온 채널의 단지 규정에 있는 액체 흐름과 같은 전기 생리적인 구체에 있는 중요한 질문에 대답하는 것을 도울 수 있습니다. 이 기술의 주요 장점은 이온 채널의 유체 흐름 조절과 관련된 데이터 해석을 위해 멤브레인 표면의 교반되지 않은 경계 층에서 실제 이온 농도를 추정할 수 있다는 것이다. 이 절차를 시작하려면 발사 유리 모세관 튜브를 구부려 U 모양을 ?...

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