Cell/Cytoplasmic Membrane Potential Activity & Analysis

Cell and/or cytoplasmic membrane potential (MP) is the electric charge inside and outside of the cell, separated by a membrane. The MP is one of the factors that determines the net flow of ions that will travel across an open ionic channel, along with the differences in concentrations between the intracellular and extracellular space.

Ions flow across the cell membrane depending on their charge; positive ions are attracted to the negative side of the membrane and vice versa. Generally, the intracellular space of cells and organelles are negative in relation to the external medium. This means electrical forces will guide the flow of positively charged ions (cations) like sodium (Na+), potassium (K+), and calcium (Ca2+) into the cell, and escort negatively charged ions (anions) like chloride (Cl-) out. In the case that there is no net ion current, the excitable membrane is considered stable.

Only live cells can maintain MP, making MP one of the most commonly used parameters to determine cell viability as it represents the differential distribution of ions across a biological membrane.

• Hyperpolarization is the shift in a cell's MP to a more negative value, which correlates to an increase in transmembrane potential that generally insinuates an increase in cell activity.
• Depolarization is the opposite effect, correlating to a decrease in transmembrane potential and cell activity, although it does not always imply cell death.

MP plays a major role in the external stimulation of the cell and ion transport across the membrane influences transmembrane nutrient transport, as well as signal transduction. In eukaryotic cells MP is not only significant to the cytoplasm, but is also important to other organelles including the mitochondria and lysosome.

 

Significance of Membrane Potential in Various Cell Types

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Bacterial Cells

In bacterial cells, membrane transport, ATP synthesis, motility, intercellular communication, and antibiotic resistance are all processes driven by ion motive forces, i.e. the MP. Bacterial cells utilize MP in many ways, including to signal and process information. By depolarizing the MP, bacteria can enter a persistent state where they become more resistant to antibiotics. Homeostasis of pH is also controlled by proton pumps to maintain a proton motive force, which is dependent on the MP.

Cell division requires MP for identifying the correct location of the division site, which may be inhibited by native protonophores. Due to the central, dynamic, role MP plays in bacteria, the bacterial cytoplasmic membrane is a major target for antimicrobial compounds like membrane-targeting peptides. Disrupting membrane function, can therefore, simultaneously inhibit one, or several of these essential processes.

 

 

Common Methods of Membrane Potential Analysis

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Fluorescent and Ratiometric Dyes

Treating cell samples with voltage-dependent fluorescent or ratiometric dyes are the most common ways of assessing cellular MP activity. Nernstian dyes are usually used to assess MP activity in regards to investigating bacteria and microbe content or contamination. They can also be used for quantitative biophysical investigation, though this requires more careful experimental consideration. Nernstian dyes can be ratiometric or fluorometric, may be cationic or anionic, can diffuse across a biological membrane, and will follow a stated behavior (calculated using the Nernst equation).

Fluorescent microscopy can be performed at different stages of the experiment, specifically before and after the introduction of the dye. The electric potential difference across the plasma membrane can be calculated (before and after dye addition) using gas constant, temperature, valence of the charged molecule, Faraday's constant, with external and internal concentrations of the charged molecule. The Nernstian dye method does possess some drawbacks as permeabilities tend to be lower with gram-negative bacteria, and these studies may require a treatment with EDTA to chelate divalent cations. Low permeability also means this method is not suitable for capturing fast dynamics. When used at high concentrations, Nernstian dyes can also become invasive to cellular MP.

ANEP dyes are another common fluorescent dye that respond to electrical potential changes. They are ultra fast-responding, and can provide millisecond response time. ANEP dyes function by responding to electric changes in their environment. These dyes can essentially shift their excitation spectra according to MP in response to electrical changes in the microenvironment. When the surrounding electric field shifts, ANEP dyes will alter their electronic structure and exhibit consequently fluorescent properties. In addition to other studies, ANEP dyes have been documented for use in MP activity analysis for single neurons, cardiac cells, intact brains, and in studying bacteria. This dye method, however, suffers from a number of drawbacks, including a weak signal-to-noise ratio. Additionally, the magnitude of their potential-dependent fluorescence changes frequently, as this measurement is dependent upon MP, which can make analysis and true quantification tricky.

Flow Cytometry

Flow cytometry has become more readily used to estimate MP in eukaryotic cells in vitro, mitochondria in vitro and in situ, and in bacteria. Flow dyes are normally non-cytotoxic, do not block ion channels, and are not extruded by the glycoprotein efflux pump. A general protocol for use is simple: cells, probes, and reagents are treated, mixed, and incubated accordingly. Cells can then be visualized and quantified by using a flow cytometer, and statistical software can be used for analysis. Flow offers many advantages as testing is simple and relatively inexpensive, result times are rapid, and subpopulation of cells or an entire sample of cells as a whole can be quantified in parallel.

Patch Clamp

Patch-clamp electrophysiology can evaluate single ion channel conductance. In this method, first a microelectrode is sealed to a cell membrane and a patch of the cell membrane containing the ion channel of interest is either disrupted or removed. After a specified voltage is applied, the voltage forms a clamp, and membrane current, rather MP, may be measured. Patch-clamp methods are generally a less-frequently used technique in research, as they are highly invasive to cells, and can only measure one cell at a time.

Application Notes:	FAQs:
Dendritic Calcium Activity Precedes Inspiratory Bursts in pre-Bötzinger Complex Neurons Neurons in lamina of mouse superior colliculus are selective for direction What's A Ratiometric Indicator	How do your membrane potential assays work? What is the difference between membrane potential and action potential? What is the difference between membrane potential and equilibrium potential?

Product Ordering Information

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Resources

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The Microbiologist's Guide to Membrane Potential Dynamics
Analysis of Antimicrobial-Triggered Membrane Depolarization Using Voltage Sensitive Dyes
The membrane potential
Cell membrane potential analysis.
Membrane Potential
Neuroanatomy, Neuron Action Potential