Intracellular pH Measurement


pH is a profound regulator of cellular function. It is, therefore, often important to assess intracellular pH. Given the small size of individual cells and their sensitivity to perturbation, measurement of intracellular pH requires sensitive, indirect measurement approaches. These include equilibration of weak acids/bases, nuclear magnetic resonance spectroscopy, pH microelectrodes, fluorescent pH indicator dyes and pH‐sensitive fluorescent proteins. Presently, the use of fluorescence techniques predominates, as these permit sensitive detection and the possibility to discretely measure pH in different cellular compartments. The selection of intracellular pH measurement technique is guided by consideration of their strengths and weaknesses, in addition to technical considerations. Cells resist changes of pH through pH buffering molecules, including proteins and bicarbonate, which together are called the cell's buffer capacity.

Key Concepts

  • Cellular processes are highly sensitive to pH, so cells have redundant mechanisms to control their pH.
  • Buffer capacity is the ability of cells to control pH by absorbing or releasing H+ from chemical pH buffering molecules.
  • Cell membranes contain embedded transport proteins able to move H+, or pH‐buffering HCO3 in order to tightly control cytosolic and organellar pH.
  • pH of the cytosol and other intracellular compartments can be measured.
  • Certain molecules will absorb light and release a photon of light at a longer wavelength, a process known as fluorescence.
  • Fluorescent dyes and proteins are the most common means to report on intracellular pH.

Keywords: cytosolic pH; organellar pH; pH indicator dye; fluorescent protein; nuclear magnetic resonance; ionophore; buffer capacity; microelectrode; fluorescence spectroscopy

Figure 1. Manipulation of intracellular pH by weak acids. Propionic acid exists as an equilibrium mixture of its protonated and deprotonated forms. While the protonated form freely diffuses across the plasma membrane, the deprotonated form, which is negatively charged, cannot diffuse across the membrane. Upon addition of propionic acid to the cell medium, protonated propionic acid diffuses into the cell until the concentration of protonated acid on both sides of the membrane is at equilibrium (their concentrations are identical). Inside the cell, an equilibrium will be established between the two forms of propionic acid, which will depend on the pH of the cytosol. All together, addition of propionic acid will cause cell pH to fall. Subsequently, removal of propionic acid from the extracellular medium leads to its diffusion of protonated propionic acid out of the cell, resulting in cellular alkalinisation as H+ are removed from the cell along with propionic acid. HA and A represent the protonated and deprotonated forms of the weak acid, respectively.
Figure 2. Mechanism of BCECF loading into the cell. In the upper part of the figure is the structure of BCECF‐AM, a membrane‐permeant derivative of BCECF, which has three carboxylic acid groups esterified with acetoxymethyl groups. After diffusion through the lipid bilayer, BCECF‐AM is acted upon by cytosolic esterases, which remove the acetoxymethyl groups, rendering the free acid negatively charged and thus unable to diffuse out of the cell. Fluorescence measurements are made by illuminating the dye at its excitation wavelength (λex) and measuring fluorescent light released at its emission wavelength (λem).
Figure 3. pH‐dependent excitation and emission spectra of BCECF. This is a diagrammatic representation of the form of data collected when fluorescence light release of BCEBF is measured at a fixed emission wavelength (λem) and the wavelength of illuminating exciting light is varied. Clearly, the amount of fluorescence released at the peak wavelength of emission (λem) increases with higher pH values. The isosbestic point is the point that all the curves pass through. At this wavelength, fluorescence emission is independent of pH.


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Further Reading

Bencina M (2013) Illumination of the spatial order of intracellular pH by genetically encoded pH‐sensitive sensors. Sensors (Basel) 13: 16736–16758.

Casey JR, Orlowski J and Grinstein S (2010) Sensors And Regulators of the Intracellular pH. Nature Reviews. Molecular Cell Biology 11: 50–61.

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Loiselle FB and Casey JR (2010) Measurement of Intracellular pH. Methods in Molecular Biology 637: 311–331.

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Malhotra, Darpan, and Casey, Joseph R(Feb 2015) Intracellular pH Measurement. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002643.pub3]