A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer.

Chon Lok Lei,Alan Fabbri,Adam P Hill,Michael Clerx,Dominic G Whittaker,Teun P De Boer,Gary R Mirams,Monique J Windley

doi:10.12688/wellcomeopenres.15968.2

Abstract

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F - containing internal solution together with a Ca 2+ containing external solution - so that a CaF 2 crystal forms when the two solutions meet which 'plugs the holes' to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F - ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Highlights

We conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current
Voltage-clamp experiments were repeated in manual patch with silicone elastomer (Figure 1C)
Measurement I The recorded leak current for Measurement I is shown in Figure 4I, which was measured with the same solutions as in Figure 2 but with silicone elastomer

Summary

Introduction

We conclude fluoride ions interacting with the CaF2 crystal are the origin of the nonlinear time-dependent leak current. Author roles: Lei CL: Conceptualization, Investigation, Methodology, Software, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing; Fabbri A: Investigation, Writing – Review & Editing; Whittaker DG: Investigation, Methodology, Writing – Review & Editing; Clerx M: Methodology, Software, Supervision, Writing – Review & Editing; Windley MJ: Investigation, Writing – Review & Editing; Hill AP: Conceptualization, Funding Acquisition, Supervision, Writing – Review & Editing; Mirams GR: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Supervision, Writing – Review & Editing; de Boer TP: Conceptualization, Funding Acquisition, Investigation, Methodology, Supervision, Writing – Review & Editing

Methods

Results

Discussion

Conclusion