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

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

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 G 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.

Citation

Lei, C. L., Fabbri, A., Whittaker, D. G., Clerx, M., Windley, M. J., Hill, A. P., …de Boer, T. P. (2021). A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer. Wellcome Open Research, 5, 1-34. https://doi.org/10.12688/wellcomeopenres.15968.2

• Journal Article Type: Article
• Acceptance Date: Jun 10, 2020
• Online Publication Date: Nov 2, 2021
• Publication Date: Nov 2, 2021
• Deposit Date: Dec 22, 2021
• Publicly Available Date: Dec 23, 2021
• Journal: Wellcome Open Research
• Electronic ISSN: 2398-502X
• Publisher: F1000 Research Ltd
• Peer Reviewed: Peer Reviewed
• Volume: 5
• Article Number: 152
• Pages: 1-34
• DOI: https://doi.org/10.12688/wellcomeopenres.15968.2
• Public URL: https://nottingham-repository.worktribe.com/output/4747510
• Publisher URL: https://wellcomeopenresearch.org/articles/5-152
• Additional Information: Referee status: Awaiting Peer Review; Grant Information: This work was supported by the Wellcome Trust through a Senior Research Fellowship to GRM [212203].; ; This work was also supported by the Engineering and Physical Sciences Research Council and the Medical Research Council [EP/L016044/1]; the Biotechnology and Biological Sciences Research Council [BB/P010008/1]; the Australian Research Council [DP190101758]; and the ZonMW MKMD programme [114021501 and 114022502]. CLL acknowledges support from the Clarendon Scholarship Fund; and the EPSRC, MRC and F. HoffmannLa Roche Ltd. for studentship support. GRM and DGW acknowledge support from the Wellcome Trust via a Wellcome Trust Senior Research Fellowship to GRM. MC acknowledges support from a BBSRC project grant. APH and MJW acknowledge support from Victor Chang Cardiovascular Innovation Centre and ARC. AF and TPdB acknowledge support from the ZonMW MKMD programme. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.; Copyright: This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.