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

Summary

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.

Abstract

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.

Introduction

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 development^1,2,3,4,5. 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 responses^5,6,7,8. For example, activation of the endothelial inward rectifier Kir2.1 and Ca²⁺-activated K⁺ (K_Ca2.3, KCNN3) channels after Ca²⁺ influx by fluid flow has been suggested to contribute to fluid flow-induced vasodilation^6,7,8. 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 technique^6,9,10,11. 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 erroneous^10,11.

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^-↔ AgCl + electron (e^-)           (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 layer^12,13,14,15) 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 (> 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 colleagues^12,13,14,15 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 current^{10,12,13,14,15}.

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.

Access restricted. Please log in or start a trial to view this content.

Protocol

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.

1. Formation of bridges
   1. 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.
2. Preparation of agarose 3 M KCl solution
   1. Prepare 100 mL of 3 M KCl solution (1 M or 2 M is also acceptable).
   2. Weigh 3 g of agarose.
   3. Dissolve the agarose in 100 mL of KCl (i.e., 3% agarose) on a hot plate between 90 and 100 °C.
3. Loading the bridges with 3 M KCl agarose
   1. 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.
   2. Keep them overnight at room temperature (RT) for the agarose to set and harden.
   3. Carefully dig out the agarose-KCl-loaded glass bridges from the set/hardened agarose-salt.
4. Storing the bridges
   1. Prepare enough volume (i.e., 500 mL) of the 3 M KCl solution in a wide-necked bottle.
   2. Store the prepared agarose-salt bridges in the bottle in a refrigerator.

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.

1. Place a container loaded with bathing solution (volume and height should already be measured) above the patch-clamp chamber.
2. Fill the patch-clamp chamber with the bathing solution by suctioning the tube.
3. 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.
4. To apply fluid flow shear force, open both tubes on the container and suction sides at the same time.
5. Before or after applying the fluid flow shear force to the cell, measure the flow rate in mL/min.
6. Calculate the flow rate by measuring the decrease in fluid volume over a given time.
7. 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).
8. 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.

3. Measuring Changes in Liquid-Metal Junction Potential by Fluid Flow Between Bath Solution and Ag/AgCl Reference Electrode (Figure 3A)

1. Use the Ag/AgCl electrode or pellet, which is available from the ready-made products, without the agarose salt bridge.
2. 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).
3. 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.
4. Fix the voltage-clamp amplifier to the current clamp mode (“I = 0” or “CC”).
5. After nullifying the initial offset potential, measure changes in voltage induced by various flow rates.
6. 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.

4. Experimental Estimation of Real Cl^- Concentration in the Unstirred Layer Adjacent to Ag/AgCl Electrode Under Static Condition (Figure 3B)

1. 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.
2. Prepare solutions with various concentrations of Cl (i.e., 50, 99, 147, 195, and 288 mM of NaCl).
3. 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 (> 30 mL/min) to prevent the decrease of Cl^- concentration to that of the adjacent Ag/AgCl reference electrode.
4. From the two relationship curves, estimate the changes in Cl^- concentration from the measured junction potential shift.

Access restricted. Please log in or start a trial to view this content.

Results

Whole cell voltage-dependent L-type Ca²⁺ channel (VDCC_L) 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 VDCC_L^11,16....

Access restricted. Please log in or start a trial to view this content.

Discussion

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...

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
RC-11 open bath chamber	Warner instruments, USA	W4 64-0307
Ag/AgCl electrode pellet	World Precision Instruments, USA	EP1
Agarose	Sigma-aldrich, USA	A9793
Voltage-clamp amplifier	HEKA, Germany	EPC8
Voltage-clamp amplifier	Molecular Devices, USA	Axopatch 200B
Liquid pump	KNF Flodos, Switzerland	FEM08