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

Abstract

Statement:We describe simplified roadmap to study the role of bioelectricity, in particular individual ions, in cell biological processes. Abstract:All cells possess an electric potential across their plasma membranes. While familiar in the context of excitable cells such as neurons, healthy non-excitable cells are also able to generate and receive bioelectric signals. The cellular resting membrane potential (RMP) regulates many factors in cell homeostasis, such as cell proliferation, differentiation and apoptosis. It is therefore critical to develop simple strategies to measure, manipulate and characterize this feature. Current studies to evaluate RMP rely on the patch clamp approach, which is technically challenging, low-throughput and not widely available to the scientific community. Here, we present a relatively simple methodology to functionally study the role of RMP in non-excitable cells by modulating it pharmacologically, and using a voltage-sensitive dye to characterize the contribution of individual ions to the RMP. Specifically, we define protocols for using extracellular solutions in which permeable ions (Na + , Cland K + ) are substituted with non-permeable ions (N-Methyl-D-glucamine (NMDG), gluconate, choline, SO4 2-) to study and manipulate RMP in vitro. 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 determine the relative contribution of ionic species in setting the RMP and be used to actively and selectively modify the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community by enabling functional modulation of RMP in most cellular assays.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential

“…In other words, membrane depolarization (|V m | decreases) corresponds to the increased fluorescence intensity. 50,54 To capture the impact of the cholesterol removal on the cell membrane potential, MBCD with different concentrations (0, 2.5, and 3 mM) and DiBAC 4 (3) probes were treated to HeLa cells. MBCD with higher concentration will remove more cholesterols from the cell membrane.…”

“…Enhanced DiBAC 4 (3) fluorescence indicates increased membrane depolarization and vice versa. 50 To remove cell membrane cholesterol, MBCD (Sigma-Aldrich, USA) was used for the extraction. 51 MBCD with different amounts was dissolved in a medium supplemented with 25 mM HEPES, which was mixed with the DiBAC 4 (3)-containing cell culture medium (2 × 10 6 cells/mL) at 1:1 ratio.…”