The proton motive force determines Escherichia coli's robustness to extracellular pH

Maintaining intracellular homeostases is a hallmark of life, and key physiological variables, such as cytoplasmic pH, osmotic pressure, and proton motive force (PMF), are typically interdependent. Developing a mathematical model focused on these links, we predict that Escherichia coli uses proton-ion antiporters to generate an out-of-equilibrium plasma membrane potential and so maintain the PMF at the constant levels observed. The strength of the PMF consequently determines the range of extracellular pH over which the cell is able to preserve its near neutral cytoplasmic pH. In support, we concurrently measure the PMF and cytoplasmic pH in single cells and demonstrate both that decreasing the PMF's strength impairs E. coli's ability to maintain its pH and that artificially collapsing the PMF destroys the out-of-equilibrium plasma membrane potential. We further predict the observed ranges of extracellular pH for which three of E. coli's antiporters are expressed, through defining their cost by the rate at which they divert imported protons from generating ATP. Taken together, our results suggest a new perspective on bacterial electrophysiology, where cells regulate the plasma membrane potential by changing the activities of antiporters to maintain both the PMF and cytoplasmic pH.

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Real-time detection of changes in yeast plasma membrane potential using genetically encoded voltage indicator proteins

ABSTRACT In yeast, adaptation to varying conditions often requires proper regulation of the plasma membrane potential. To determine yeast membrane potential change, optical methods involving potentiometric dyes have been supplemental to the direct electrode-based method. However, the hydrophobic nature of the dyes and their slow distribution across the membrane still limits their utilization. Genetically encoded voltage indicator (GEVI) proteins employed in neuroscience offer a tantalizing alternative for monitoring yeast membrane potential change. In this work, several widely used GEVI proteins were assessed in Saccharomyces cerevisiae for their expression and function as a voltage reporter. Among them, only ArcLight and Accelerated Sensor of Action Potential (ASAP) proteins could be expressed and transported to the plasma membrane. While the voltage-sensing capability was demonstrated for both ArcLight and ASAP, ArcLight fluorescence was sensitive to the intracellular pH change concurrently with the voltage change. Therefore, we established that ASAP is the more suitable GEVI protein for reporting yeast membrane potential change. This voltage-sensing reporter for yeast based on ASAP offers a new effective strategy for real-time optical detection of yeast membrane potential change, which potentially facilitates many areas of yeast research including optimizing growth conditions for industrial use and investigating yeast ion transport system.