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Intracellular pH (pHi) is the measure of the acidity or basicity (i.e., pH) of intracellular fluid. The pHi plays a critical role in membrane transport and other intracellular processes. In an environment with the improper pHi, biological cells may have compromised function.<ref>Template:Cite journal</ref><ref>Template:Cite journal</ref> Therefore, pHi is closely regulated in order to ensure proper cellular function, controlled cell growth, and normal cellular processes.<ref name=":0" /> The mechanisms that regulate pHi are usually considered to be plasma membrane transporters of which two main types exist — those that are dependent and those that are independent of the concentration of bicarbonate (Template:Chem). Physiologically normal intracellular pH is most commonly between 7.0 and 7.4, though there is variability between tissues (e.g., mammalian skeletal muscle tends to have a pHi of 6.8–7.1).<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref> There is also pH variation across different organelles, which can span from around 4.5 to 8.0.<ref name=":2">Template:Cite journal</ref><ref>Template:Cite journal</ref> pHi can be measured in a number of different ways.<ref name=":0">Template:Cite journal </ref><ref>Template:Cite journal</ref>
HomeostasisEdit
Intracellular pH is typically lower than extracellular pH due to lower concentrations of HCO3−.<ref>Template:Cite journal Template:Verify source</ref> A rise of extracellular (e.g., serum) partial pressure of carbon dioxide (pCO2) above 45 mmHg leads to formation of carbonic acid, which causes a decrease of pHi as it dissociates:<ref>Flinck M, Kramer SH, Pedersen SF (July 2018). "Roles of pH in control of cell proliferation". Acta Physiol (Oxf). 223 (3): e13068. doi:10.1111/apha.13068. PMID 29575508.</ref>
- H2O + CO2 Template:Eqm H2CO3 Template:Eqm H+ + HCO3–
Since biological cells contain fluid that can act as a buffer, pHi can be maintained fairly well within a certain range.<ref>Template:Cite journal</ref> Cells adjust their pHi accordingly upon an increase in acidity or basicity, usually with the help of CO2 or HCO3– sensors present in the membrane of the cell.<ref name=":0" /> These sensors can permit H+ to pass through the cell membrane accordingly, allowing for pHi to be interrelated with extracellular pH in this respect.<ref>Template:Cite journal</ref>
Major intracellular buffer systems include those involving proteins or phosphates. Since the proteins have acidic and basic regions, they can serve as both proton donors or acceptors in order to maintain a relatively stable intracellular pH. In the case of a phosphate buffer, substantial quantities of weak acid and conjugate weak base (H2PO4– and HPO42–) can accept or donate protons accordingly in order to conserve intracellular pH:<ref>{{#invoke:citation/CS1|citation |CitationClass=web }}</ref><ref>Template:Cite journal</ref>
- OH– + H2PO4– Template:Eqm H2O + HPO42–
- H+ + HPO42– Template:Eqm H2PO4–
In organellesEdit
The pH within a particular organelle is tailored for its specific function.
For example, lysosomes have a relatively low pH of 4.5.<ref name=":2" /> Additionally, fluorescence microscopy techniques have indicated that phagosomes also have a relatively low internal pH.<ref name=":4">Template:Cite journal</ref> Since these are both degradative organelles that engulf and break down other substances, they require high internal acidity in order to successfully perform their intended function.<ref name=":4" />
In contrast to the relatively low pH inside lysosomes and phagosomes, the mitochondrial matrix has an internal pH of around 8.0, which is approximately 0.9 pH units higher than that of inside intermembrane space.<ref name=":2" /><ref>Template:Cite journal</ref> Since oxidative phosphorylation must occur inside the mitochondria, this pH discrepancy is necessary to create a gradient across the membrane. This membrane potential is ultimately what allows for the mitochondria to generate large quantities of ATP.<ref>Template:Cite book</ref>
MeasurementEdit
There are several common ways in which intracellular pH (pHi) can be measured including with a microelectrode, dye that is sensitive to pH, or with nuclear magnetic resonance techniques.<ref name=":3">Template:Cite journal</ref><ref name=":1" /> For measuring pH inside of organelles, a technique utilizing pH-sensitive green fluorescent proteins (GFPs) may be used.<ref name=":5">Template:Cite journal</ref>
Overall, all three methods have their own advantages and disadvantages. Using dyes is perhaps the easiest and fairly precise, while NMR presents the challenge of being relatively less precise.<ref name=":3" /> Furthermore, using a microelectrode may be challenging in situations where the cells are too small, or the intactness of the cell membrane should remain undisturbed.<ref name=":1" /> GFPs are unique in that they provide a noninvasive way of determining pH inside different organelles, yet this method is not the most quantitatively precise way of determining pH.<ref name=":6" />
MicroelectrodeEdit
The microelectrode method for measuring pHi consists of placing a very small electrode into the cell’s cytosol by making a very small hole in the plasma membrane of the cell.<ref name=":1" /> Since the microelectrode has fluid with a high H+ concentration inside, relative to the outside of the electrode, there is a potential created due to the pH discrepancy between the inside and outside of the electrode.<ref name=":3" /><ref name=":1" /> From this voltage difference, and a predetermined pH for the fluid inside the electrode, one can determine the intracellular pH (pHi) of the cell of interest.<ref name=":1">Template:Cite book </ref>
Fluorescence spectroscopyEdit
Another way to measure Intracellular pH (pHi) is with dyes that are sensitive to pH, and fluoresce differently at various pH values.<ref name=":4" /><ref>Template:Cite journal</ref> This technique, which makes use of fluorescence spectroscopy, consists of adding this special dye to the cytosol of a cell.<ref name=":3" /><ref name=":1" /> By exciting the dye in the cell with energy from light, and measuring the wavelength of light released by the photon as it returns to its native energy state, one can determine the type of dye present, and relate that to the intracellular pH of the given cell.<ref name=":3" /><ref name=":1" />
Nuclear magnetic resonanceEdit
In addition to using pH-sensitive electrodes and dyes to measure pHi, Nuclear Magnetic Resonance (NMR) spectroscopy can also be used to quantify pHi.<ref name=":1" /> NMR, typically speaking, reveals information about the inside of a cell by placing the cell in an environment with a potent magnetic field.<ref name=":3" /><ref name=":1" /> Based on the ratio between the concentrations of protonated, compared to deprotonated, forms of phosphate compounds in a given cell, the internal pH of the cell can be determined.<ref name=":3" /> Additionally, NMR may also be used to reveal the presence of intracellular sodium, which can also provide information about the pHi.<ref>Template:Cite journal</ref>
Using NMR Spectroscopy, it has been determined that lymphocytes maintain a constant internal pH of 7.17± 0.06, though, like all cells, the intracellular pH changes in the same direction as extracellular pH.<ref>Template:Cite journal</ref>
pH-sensitive GFPsEdit
To determine the pH inside organelles, pH-sensitive GFPs are often used as part of a noninvasive and effective technique.<ref name=":5" /> By using cDNA as a template along with the appropriate primers, the GFP gene can be expressed in the cytosol, and the proteins produced can target specific regions within the cell, such as the mitochondria, golgi apparatus, cytoplasm, and endoplasmic reticulum.<ref name=":6">Template:Cite journal</ref> If certain GFP mutants that are highly sensitive to pH in intracellular environments are used in these experiments, the relative amount of resulting fluorescence can reveal the approximate surrounding pH.<ref name=":6" /><ref>Template:Cite journal</ref>