There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp...

Area of Science:

• Electrophysiology
• Ion Channel Research
• Pharmacology

Background:

• Fluoride-containing internal solutions are standard in patch clamp electrophysiology for enhancing seal resistance and recording duration.
• Fluoride's presence can interfere with ion channel gating kinetics and intracellular signaling pathways, necessitating fluoride-free alternatives.
• Automated patch clamp (APC) systems rely on internal fluoride to achieve high gigaseal (GΩ) formation rates, crucial for data quality.

Purpose of the Study:

• To develop and validate a high-throughput, fluoride-free internal solution for automated patch clamp experiments.
• To assess the efficacy of the fluoride-free approach in achieving high GΩ seal rates across various cell lines and expressed ion channels.
• To provide a comprehensive comparison of fluoride-containing versus fluoride-free solutions in patch clamp electrophysiology.

Main Methods:

• Development of a novel, high-throughput, fluoride-free internal recording solution.
• Implementation on a 384-well based automated patch clamp system.
• Validation using cells expressing human ether-à-go-go-related gene (hERG), sodium channel Na_V1.5, Na_V1.7, and potassium calcium-activated channel K_Ca3.1.

Main Results:

• Achieved success rates exceeding 40% for gigaseal (GΩ) formation using the fluoride-free internal solution in a high-throughput APC system.
• Successfully recorded from multiple ion channels including hERG, Na_V1.5, Na_V1.7, and K_Ca3.1 expressed in HEK and CHO cells.
• Demonstrated the feasibility of performing patch clamp experiments without internal fluoride, preserving physiological channel behavior.

Conclusions:

• The developed fluoride-free internal solution offers a viable and effective alternative for automated patch clamp recordings.
• This approach overcomes the limitations of fluoride, such as altered channel gating and second messenger system interference.
• The method is advantageous for studying ion channels where precise voltage-dependence and physiological intracellular conditions are critical.