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 symptoms such as tinnitus, hearing loss, and other related issues. These symptoms are frequently associated with conditions such as cervical spondylosis, cerebral atherosclerosis, and abnormal blood pressure. Cerebrovascular disease, particularly prevalent among middle-aged and elderly individuals, is often linked to these underlying conditions^2,3,4.

Resistance arteries play a vital role in cardiovascular function and maintaining bodily homeostasis. As the primary site of vascular resistance, they regulate blood pressure and cardiac output, ensuring sufficient blood flow to meet the metabolic and physiological demands of tissues and organs⁵. The basilar artery, classified as a resistance artery, primarily regulates blood flow to the brainstem⁶. Smooth muscle cells, which form the walls of resistance arteries, are key mediators of vascular resistance through the regulation of steady-state contraction or vascular tension. These cells harbor numerous ion channels, including K⁺ channels, Ca²⁺ channels, and Cl^- channels, which are critical for the modulation of vascular tone^5,7.

K⁺ channels are critical in establishing the membrane potential and regulating the contractile tone of arterial smooth muscle cells⁸. There are four types of K⁺ channels in arterial smooth muscle: voltage-dependent K⁺ (Kᴠ), Ca²⁺-dependent K⁺ (K_Ca), ATP-dependent K⁺ (K_ATP), and inward rectifier K⁺ (Kir) channels^9,10,11. Kir channels are categorized into seven subtypes, with Kir2.x being classical Kir channels. Among these, the Kir2.x subfamilies are the most relevant in the vasculature. Kir currents exhibit inward rectification at negative voltages, indicating a net influx of K⁺ into the cell, whereas at positive voltages, there is minimal to no net K⁺ current flow⁵. In the cardiovascular system, Kir channels are essential for stabilizing the membrane potential. Their activation induces cell membrane hyperpolarization and vasodilation^12,13,14.

Patch-clamp experiments on freshly isolated smooth muscle cells have been conducted in various arteries, including coronary, cerebral, renal, and mesenteric arteries^15,16. While some methods utilize the same type of collagenase for cell isolation, the precise procedures vary. Few studies have comprehensively summarized the methods for isolating vascular smooth muscle cells. Therefore, this study focuses on the fresh isolation of primary vascular smooth muscle cells from the rat basilar artery and the recording of Kir channel currents in these cells using the whole-cell patch clamp technique, providing a detailed and complete protocol for researchers in related fields.

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 rat by inhalation of 2% isoflurane. Confirm deep anesthesia using a toe pinch. If required, administer additional anesthetics. Immediately proceed to open the skull and expose the brain on the portable operating table.
3. Quickly transfer the brain to a Petri dish containing PSS saturated with 95% O₂ and 5% CO₂ at 4 °C. Ensure the solution has a pH of 7.40.
4. Position the brain ventrally upward in a Petri dish and secure it with needles. Under a light microscope, locate the basilar artery and carefully remove the surrounding tissue using autoclaved tweezers and scissors (see Figure 1A).
5. Insert a 2 cm-long wire with a diameter of 25 µm into the isolated basilar artery. Gently rub the inner wall with the wire to effectively remove the vascular endothelium.
   NOTE: The basilar artery in rats is situated within the basal sulci of the brainstem at the base of the brain and is formed by the confluence of the left and right vertebral arteries². During the isolation process, handle the artery with care to avoid excessive stretching or compression that could result in damage.

2. Isolation of smooth muscle cells

1. Preheat 1 mL of cell separation solution, 1 mL of enzymatic hydrolysate I, and 1 mL of enzymatic hydrolysate II to 37 °C in test tubes 1, 2, and 3, respectively (see Figure 1B).
2. Transfer the isolated basilar artery to test tube 2. Continuously inject a mixture of 95% O₂ and 5% CO₂ into the tube, and maintain enzymatic treatment for 30 min (see Figure 1C).
3. Transfer the basilar artery from test tube 2 to test tube 3. Continue injecting 95% O₂ and 5% CO₂ into the tube, and maintain enzymatic treatment for 5 min (see Figure 1D).
4. Add 1 mL of 37 °C cell separation solution from test tube 1 into test tube 3. Continue injecting 95% O₂ and 5% CO₂ while maintaining enzymatic treatment for 3 min. Triturate the basilar artery preparation to release cells (see Figure 1E).
5. Add 4 °C separation solution to test tube 3 to terminate the enzymatic process (see Figure 1F). Centrifuge the mixture at 59 × g for 6 min. Discard the supernatant using a pipette, add 4 °C separation solution again (see Figure 1G), and repeat centrifugation twice to remove residual enzymes.
6. Remove the supernatant using a pipette, and store 1 mL of the cell suspension at 4 °C for up to 6-8 h.
7. Take 100 µL of the cell suspension and place it in the bath (see Figure 1H). Add 1 mL of extracellular fluid to the bath and allow the cells to settle for 40 min to attach to the bottom (see Figure 2).
   NOTE: For cells in the treatment group (e.g., BaCl₂ or sodium nitroprusside), pre-incubate with the respective substances at room temperature (22-26°C) for 40 min during the attachment period.

3. Recording Kir current using whole-cell patch clamp

1. Fabrication of micropipettes
   1. Turn on the micropipette puller (refer to the Table of Materials).
   2. Place a glass tube (outer diameter: 1.5 mm, inner diameter: 1.10 mm, length: 10 cm) in the puller. Select Program 1, click on Enter, and access Program 1. Perform a ramp test by clicking on Ramp on the control panel to measure the heat value of the glass tube.
      NOTE: Edit Program 1 with the following parameters: Heat: Ramp, Pull: 0, Velocity: 25, Delay: 1, Pressure: 500, Mode: Delay, Safe heat enabled. The ramp test must be performed when changing filaments or glass pipette types. Micropipettes should have a tip diameter of ~1-2 µm and a cone length of approximately 5 mm. Smaller tip diameters and longer cones result in higher pipette resistance.
   3. Insert a new glass tube and select Program 1. Click on Enter to fabricate the micropipette.
2. Recording Kir Current
   1. Turn on the hardware devices sequentially: digital-to-analog converter, signal amplifier, micromanipulator, microscope, camera, and computer.
   2. Launch the software in the following order: camera, signal amplifier, and data acquisition software.
      NOTE: If a microscope is not equipped with a camera, only the instrument software needs activation. Ensure software is opened after hardware initialization to enable proper functionality.
   3. In the data acquisition software, select File > Set Data File Names to establish a data storage path.
   4. Edit the protocol for recording Kir currents as follows:
      1. Waveform interface: Epoch A & D: Type = step; First level = −60 mV; Delta level = 0 mV; First duration = 100 ms; Delta duration = 0 ms. Epoch B: Type = step; First level = −160 mV; Delta level = 0 mV; First duration = 1 ms; Delta duration = 0 ms. Epoch C: Type = ramp; First level = 40 mV; Delta level = 20 mV; First duration = 500 ms; Delta duration = 0 ms.
      2. Mode/Rate interface: Trial delay = 0 s; Runs = 1; Sweeps = 1; Sweep duration = 0.8 s.
      3. Outputs interface: Channel #0; Holding level = −60 mV. Save and name the protocol as "Kir protocol."
         NOTE: Protocol setup is tailored for specific current recordings and can be reused after saving. Ensure that the output interface matches the instrument's wiring (e.g., Channel #0).
   5. Enable the Membrane Test function in the Tools menu of the data acquisition software.
   6. Position the cell for measurement in the center of the microscope's camera view.
   7. Immerse the reference wire tip into the bath solution. Fill 20% of the micropipette (fabricated in step 3.1) with the intracellular solution and secure it on the recording electrode holder.
      1. Apply positive pressure using a syringe, move the pipette tip into the bath solution, and adjust the pipette offset on the signal amplifier software to set the current baseline to 0 pA (see Figure 3A).
         NOTE: Use Ag/AgCl reference wires. Pipette resistance should be 4-6 MΩ.
   8. Gently press the pipette against the cell membrane. Observe an increase in seal resistance (Rt) to at least 1 MΩ (Figure 3B). Remove positive pressure and apply negative pressure to establish a high resistance with Rt ≥ 1 GΩ (Figure 3C).
      1. Set the holding voltage to −60 mV and compensate electrode capacitance using Cp Fast and Cp Slow (Figure 3D).
         NOTE: If Rt remains above 1 GΩ, proceed to the next step; otherwise, replace the cell or pipette and repeat steps 3.2.6-3.2.8.
   9. Apply brief negative pressure to rupture the cell membrane, forming a whole-cell configuration (Figure 3E). Select Whole Cell in the signal amplifier software, click on Auto for membrane capacitance compensation (Figure 3F), load the Kir protocol, and begin data recording. Repeat steps 3.2.6-3.2.9 for treated cells.
      NOTE: If the series resistance (Ra) is >30 MΩ, select a new cell. If 30 MΩ > Ra > 10 MΩ, apply series resistance compensation before recording. This protocol requires Ra < 10 MΩ.
3. Analyzing data
   1. Open the data analysis software and load the recorded data. Use cursors to analyze current traces and export data for I-V curve plotting.
   2. Create a stimulus waveform signal by selecting Edit > Create Stimulus Waveform Signal and confirm.
   3. Export the Kir protocol trace by selecting Edit > Transfer Traces, specifying the full trace region and signal (A0 #0), and copying data for further analysis.
   4. Export representative Kir current traces by selecting Edit > Transfer Traces, specifying the full trace region and signal (IN 0), and copying data to generate current trace plots.
      NOTE: Normalize current data using current density (pA/pF) for accurate comparison between cells of varying sizes. Ensure membrane capacitance (Cm) is recorded during the experiment¹⁷.

Results

Isolation of arterial smooth muscle cells
The first section of the procedure details the process of isolating smooth muscle cells from the rat's cerebral basilar artery. This process is illustrated in Figure 1. The procedure involves enzymatic digestion and cell separation steps to release smooth muscle cells from the artery.

Representative images of isolated smooth muscle cells
The second section presents a repres...

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