Recording of Inward Rectifying K+ Currents in Freshly Isolated Basilar Artery Smooth Muscle Cells by Patch Clamp Technique

Summary

This protocol describes a rapid and efficient method for isolating smooth muscle cells from the rat basilar artery and recording inward rectifying potassium channel currents in these cells using the whole-cell patch clamp technique. It offers a novel approach for researchers studying the basilar artery and ion channels.

Abstract

Cerebrovascular disease is a prevalent condition among the elderly, with its incidence steadily rising. The basilar artery is a critical cerebral vessel that supplies the pons, cerebellum, posterior brain regions, and inner ear. Potassium (K⁺) channel activity plays a significant role in determining vascular tone by regulating the cell membrane potential. Activation of inward rectifying K⁺ (Kir) channels, like other K⁺ channels, leads to cell membrane hyperpolarization and vasodilation. In this study, freshly isolated smooth muscle cells from the basilar artery were used to record Kir currents via the whole-cell patch clamp technique. The effects of 100 µmol/L BaCl₂, a Kir channel inhibitor, and 10 µmol/L sodium nitroprusside (SNP), a nitro vasodilator, on Kir channel currents were investigated. The results demonstrated that BaCl₂ inhibited Kir channel currents in basilar artery smooth muscle cells, whereas SNP enhanced these currents. This protocol provides a comprehensive guide for preparing freshly isolated arterial smooth muscle cells and recording Kir channel currents using the patch clamp technique, offering a valuable resource for researchers seeking to master this method.

Introduction

Cerebrovascular disease is a prevalent condition in the elderly population. With improvements in living standards, increased life expectancy, and the aging population, the incidence of cerebrovascular disease is steadily rising¹. The basilar artery, an unpaired vessel formed by the fusion of the bilateral vertebral arteries, runs beneath the pons within the skull and divides into two posterior cerebral arteries. It supplies the pons, cerebellum, posterior regions of the brain, and the inner ear. Insufficient blood supply to the basilar artery can lead to episodic vertigo, often accompanied by nausea and vomiting. Patients may also experience sy....

Protocol

The animal protocol was approved by the Chengdu University of Traditional Chinese Medicine Laboratory Animal Welfare Ethics Committee (Record No. 2024035). Male Sprague-Dawley (SD) rats, weighing 260-300 g and aged 8-10 weeks, were used in this study. The animals were provided with water and food (SPF experimental animal feed) ad libitum. Details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Rat basilar artery dissection

1. Solution Preparation
   1. Prepare solutions as described in Table 1.
2. Anesthetize the ....

Discussion

Whole-cell recording using freshly isolated cells dates back to the early 1980s¹⁸, and the recording of channel currents from rodent basilar smooth muscle cells became widely practiced in the 1990s¹⁹. With technological advancements, researchers are increasingly focused on the results achieved through these technologies. However, the attention given to updating and summarizing technical methods has gradually diminished. This paper introduces a detailed method for the fresh .......

Materials

Name	Company	Catalog Number	Comments
Bovine serum albumin	Sigma, USA	B2064
Barium chloride	Macklin Biochemical Co.,Ltd.,Shanghai, China	B861682
CaCl2	Sangon Biotech Co., Ltd., Shanghai, China	A501330
Camera	Hamamatsu, Japan	C11440
Camera software	Image J, USA	Micro-manager 2.0.0-gammal
Collagenase F	Sigma, USA	C7926
Collagenase H	Sigma, USA	C8051
Computer	Lenovo, China	~
Data acquisition software	Molecular Devices, USA	Clampex 10.4
Data analysis software	Axon, USA	clampfit 10.4
D-glucose	Sangon Biotech Co., Ltd., Shanghai, China	A610219
Digital-analog converter	Molecular Devices, USA	Axon digidata 1550B
Dithiothreitol	Sigma, USA	D0632
Drawing software	San Diego, California, USA	GraphPad
EGTA	Sangon Biotech Co., Ltd., Shanghai, China	A600077
Glass tube	DL Naturegene Life Sciences.USA	B150-86-10
HEPES	Xiya Reagent Co., Ltd., Shandong, China	S3872
KCl	Sangon Biotech Co., Ltd., Shanghai, China	A100395
KH2PO4	Sangon Biotech Co., Ltd., Shanghai, China	A100781
MgCl2·6H2O	Sangon Biotech Co., Ltd., Shanghai, China	A100288
Micromanipulator	sutter, USA	MP285A
Micropipette puller	sutter, USA	P1000
Microscope	Olympus, Japan	IX73
Na2-ATP	Sigma, USA	A26209
Na2HPO4	Sangon Biotech Co., Ltd., Shanghai, China	A610404
NaCl	Sangon Biotech Co., Ltd., Shanghai, China	A100241
NaH2PO4	Sangon Biotech Co., Ltd., Shanghai, China	A600878
NaHCO3	Sangon Biotech Co., Ltd., Shanghai, China	A100865
NaOH	Sangon Biotech Co., Ltd., Shanghai, China	A100173
Papain	Sigma, USA	P4762
Potassium-D-gluconate	Sangon Biotech Co., Ltd., Shanghai, China	A507810
Signal amplifier	Molecular Devices, USA	Axon MutiClamp 700B
Signal amplifier software	Molecular Devices, USA	MultiClamp Commander software
Sodium nitroprusside	Sangon Biotech Co., Ltd., Shanghai, China	A600867
Statistical analysis software	San Diego, California, USA	GraphPad