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Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential.

ABSTRACT: All cells possess an electric potential across their plasma membranes and can generate and receive bioelectric signals. The cellular resting membrane potential (RMP) can regulate cell proliferation, differentiation and apoptosis. Current approaches to measure the RMP rely on patch clamping, which is technically challenging, low-throughput and not widely available. It is therefore critical to develop simple strategies to measure, manipulate and characterize the RMP. Here, we present a simple methodology to study the RMP of non-excitable cells and characterize the contribution of individual ions to the RMP using a voltage-sensitive dye. We define protocols using extracellular solutions in which permeable ions (Na⁺, Cl^- and K⁺) are substituted with non-permeable ions [N-Methyl-D-glucamine (NMDG), gluconate, choline, SO₄ ^2-]. The resulting RMP modifications were assessed with both patch clamp and a voltage sensitive dye. Using an epithelial and cancer cell line, we demonstrate that the proposed ionic solutions can selectively modify the RMP and help determine the relative contribution of ionic species in setting the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community.This article has an associated First Person interview with the first author of the paper.

SUBMITTER: Bonzanni M

PROVIDER: S-EPMC6994931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear.MethodsWe tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondrial membrane potential (ΔΨ m), reactive oxygen species production (ROS), and cellular membrane potential (Vm) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose-response curves, which allowed testing of ≥90 commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography-mass spectrometry.ResultsWe found that e-liquids caused a decrease in ΔΨ m and Vm and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied.ConclusionOur data suggest that ΔΨ m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.ImplicationsE-liquid cellular toxicity can be predicted using parameters amenable to HTS. Our data suggest that ΔΨ m, ROS, Vm, and toxicity may be indicative of the extent of cell death upon e-liquid exposure, and this toxicity is linked to the chemical composition, that is, flavoring components. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.

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Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential.

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