全自动膜片钳技术实验优化以及与手动膜片钳技术的对比研究——以TRPV1通道为例

doi:10.5246/jcps.2025.05.037

中国药学(英文版) ›› 2025, Vol. 34 ›› Issue (5): 485-492.DOI: 10.5246/jcps.2025.05.037

全自动膜片钳技术实验优化以及与手动膜片钳技术的对比研究——以TRPV1通道为例

段桂芳^*(), 李樾, 袁霞, 宋书香, 许迎利, 周建春

北京大学药学院天然药物及仿生药物全国重点实验室, 北京 100191

收稿日期:2025-01-04 修回日期:2025-02-20 接受日期:2025-03-07 出版日期:2025-06-02 发布日期:2025-06-01
通讯作者: 段桂芳

Optimization of automated patch-clamp technique and comparative analysis with manual patch-clamp: A case study on the TRPV1 channel

Guifang Duan^*(), Yue Li, Xia Yuan, Shuxiang Song, Yingli Xu, Jianchun Zhou

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2025-01-04 Revised:2025-02-20 Accepted:2025-03-07 Online:2025-06-02 Published:2025-06-01
Contact: Guifang Duan
Supported by:
The National Natural Science Foundation of China (Grant No. 32000674).

摘要/Abstract

摘要：

膜片钳技术能够精确测量和分析离子通道蛋白的电流特性, 为筛选和优化药物提供关键的分子层面信息, 加速药物的发现和开发过程。近年来, 全自动膜片钳技术逐渐发展, 相比于手动膜片钳具有高通量、高质量和自动化的优点。然而, 需要针对不同靶点将实验条件优化才能发挥其技术优势, 否则昂贵的耗材费用等实验成本会大大限制该技术的发展。本研究以TRPV1通道为实验对象, 介绍全自动膜片钳检测TRPV1通道电流的方法建立和参数优化, 以及与手动膜片钳技术检测结果的对比分析。

关键词: 膜片钳技术, 离子通道, 实验方法建立, 药理学

Abstract:

Patch-clamp technique serve as a powerful tool for precisely measuring and characterizing ion channel currents, offering critical molecular-level insights essential for drug screening and optimization. By enabling a deeper understanding of ion channel behavior, these techniques significantly accelerate the process of drug discovery and development. In recent years, automated patch-clamp technique has undergone substantial advancements, surpassing traditional manual methods with its high throughput, improved data consistency, and automation. However, fully harnessing these advantages requires meticulous optimization of experimental conditions tailored to specific targets. Without such refinement, the high cost of consumables and operational expenses could severely hinder the widespread adoption of this technique. This study focused on the TRPV1 channel, detailing the establishment of an automated patch-clamp detection system for TRPV1 currents, optimization of experimental parameters, and a comparative evaluation of the results against manual patch-clamp techniques.

Key words: Patch-clamp technique, Ion channels, Methods establishment, Pharmacology

Supporting:

段桂芳, 李樾, 袁霞, 宋书香, 许迎利, 周建春. 全自动膜片钳技术实验优化以及与手动膜片钳技术的对比研究——以TRPV1通道为例[J]. 中国药学(英文版), 2025, 34(5): 485-492.

Guifang Duan, Yue Li, Xia Yuan, Shuxiang Song, Yingli Xu, Jianchun Zhou. Optimization of automated patch-clamp technique and comparative analysis with manual patch-clamp: A case study on the TRPV1 channel[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(5): 485-492.

图/表 6

Figure 1. (A) The structure of the Qplate used in APC; (B) Structure of the compound; (C) Establishment of methods for detecting TRPV1 channel currents using APC techniques and the optimization of parameters.

Table 1. Qpatch protocol.

Figure 2. The success rate of APC experiments before (A) and after (B) optimization.

Figure 3. HEK293T-TRPV1 cell currents recorded on APC. (A) Raw current with increasing concentration of capsaicin and blocked by BCTC (10 μmol/L) with ISCs; (B) The I-T plot of (A); (C) Raw current with increasing concentration of capsaicin and blocked by BCTC (10 μmol/L) with ISK; (D) The I-T plot of (C); (E) Maximal outward TRPV1 currents were recorded on QPatch16 at +100 mV (in ISCs and ISK, respectively). Data are presented as mean ± SEM, **P < 0.01.

Figure 4. Whole-cell recordings were obtained from a representative HEK293T-TRPV1 cell using MPC techniques. (A) Currents were recorded in the presence of a K+-based internal solution (ISK) at +100 mV. The application of capsaicin and BCTC is indicated by horizontal bars;(B) Ramped I–V curves of TRPV1 corresponding to the currents shown in (A); (C) Currents recorded in the presence of a Cs+-based internal solution (ISCs) at +100 mV. The application of capsaicin and control buffer is indicated by horizontal bars; (D) Ramped I–V curves of TRPV1 corresponding to the currents shown in (C); (E) Maximal outward TRPV1 currents were recorded using MPC at +100 mV in ISCs and ISK conditions, respectively. Data are presented as mean ± SEM, ***P < 0.001.

Figure 5. TRPV1 current density recorded by APC and MPC in K-based and Cs-based internal solutions, respectively, at +100 mV. Data are plotted as mean ± SEM.

参考文献 14

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全自动膜片钳技术实验优化以及与手动膜片钳技术的对比研究——以TRPV1通道为例

Optimization of automated patch-clamp technique and comparative analysis with manual patch-clamp: A case study on the TRPV1 channel

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