Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Summary

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Abstract

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K⁺ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K⁺ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

Introduction

Hemodynamic forces generated by the blood flow are well known to play major roles in endothelial and vascular function^1,2. It is also well known that several types of ion channels acutely respond to changes in shear stress^3,4,5 leading to the hypothesis that ion channels can be primary shear stress sensors. More recently, we and others showed that mechanosensitive ion channels play critical roles in several shear-stress sensitive vascular functions, including the vasoactive response to shear stress^6<....

Protocol

The use of animals in our studies is approved by the University of Illinois at Chicago Animal Care Committee (#16-183).

1. Assembly of the Modified Parallel Plate Flow Chamber

NOTE: Please refer to Table 1 and Figure 1 for MPP flow chamber piece IDs. Please refer to Figure 1 for a schematic detailing the orientation of chamber pieces for assembly.

1. To adhere the .......

Discussion

The vascular system is constantly exposed to active hemodynamic forces, which activate mechanosensitive ion channels^3,22 but the physiological roles of these channels in shear stress-induced mechanotransduction is only starting to emerge^4,6,8. The mechanisms responsible for the mechanosensitivity of shear stress-activated channels remain unknown. The protocol detailed he.......

Materials

Name	Company	Catalog Number	Comments
0.2 µm sterile syringe filters	VWR	28145-501	Used for filtering electrophysiolgoical pipette solution
5 grade forceps	Fine Scientific Tools	1252-30	Used for transferring digested arteries to fresh solution
9" Pasteur Pipet	Fisher Scientifc	13-678-20D	Used for mechanically disrupting digested arteries and transferring freshly isolated endohtelial cells
12 mm diameter Cover glass circles	Fisher Scientifc	12-545-80	For use with studies involving cultured cells and multiple treatments. Cells adhered to the cover glass are used for patch clamp analyses
24 x 40 mm Rectangluar Cover glass	Sigma-Aldrich	CLS2975224	Cover glass to be added to MPP flow chamber pieces C (Figure 1)
24 x 50 mm Rectangluar Cover glass	Sigma-Aldrich	CLS2975245	Cover glass to be added to MPP flow chamber E (Figure 1)
20 gauge syringe needles	Becton Dickinson and Co	305175	For use in mechanical disruption of digested mesenteric arteries
35 mm Petri dish	Genesee Scientific	32-103	For use in mechanical disruption of digested mesenteric arteries
Amphotericin B solubilized	Sigma-Aldrich	A9528-50MG	Used for generating the perforated whole-cell patch configuration.
collagenase, type I	Worthington Biochemical	100 mg - LS004194	Enzyme used in our laboratory as a brief digestion following the initial cocktail of neutral protease and elastase
Dimethyl Sulfoxide (DMSO)	Fisher Scientifc	67-68-5	Solvent for Amphotericin B used in perforated whole-cell patch clamp
elastase, lyophilized	Worthington Biochemical	25 mg - LS002290	Enzyme used in our laboratory in a cocktail with neutral protease/dispase to begin digestion of arteries for endothelial cell isolation.
Falcon Tissue culture Plate, 6-well, Flat Bottom with Low Evaporation Lid	Corning	353046	For use with studies involving cultured cells and multiple treatments
neutral protease/dispase	Worthington Biochemical	10 mg- LS02100 50 mg - LS02104	Enzyme used in our laboratory in a cocktail with elastase to begin digestion of arteries for endothelial cell isolation
SylGard	World Precision Instruments	SYLG184	Silicone elastomer for adhering the rectangular cover slip to the MPP flow chamber pieces C and E (Figure 1)
Tygon ND 10-80 tubing	Microbore Tubing	AAQ04133	ID: 0.05 in, OD: 0.09 in, inlet perfusion tubing for adminsitering flow to the chamber