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,

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.
   ....

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........

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.......

Materials

List of materials used in this article

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