Intracellular pH

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pH gradient across a membrane, with protons traveling through a transporter embedded in the membrane. Membrane pH gradient.jpg
pH gradient across a membrane, with protons traveling through a transporter embedded in the membrane.

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. [1] [2] Therefore, pHi is closely regulated in order to ensure proper cellular function, controlled cell growth, and normal cellular processes. [3] 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 (HCO
3
). 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). [4] [5] There is also pH variation across different organelles, which can span from around 4.5 to 8.0. [6] [7] pHi can be measured in a number of different ways. [3] [8]

Contents

Homeostasis

Intracellular pH is typically lower than extracellular pH due to lower concentrations of HCO3. [9] 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: [10]

H2O + CO2 H2CO3 H+ + HCO3

Since biological cells contain fluid that can act as a buffer, pHi can be maintained fairly well within a certain range. [11] 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. [3] These sensors can permit H+ to pass through the cell membrane accordingly, allowing for pHi to be interrelated with extracellular pH in this respect. [12]

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: [13] [14]

OH + H2PO4 H2O + HPO42–
H+ + HPO42– H2PO4

In organelles

Approximate pHs of various organelles within a cell. PH of organelles.jpg
Approximate pHs of various organelles within a cell.

The pH within a particular organelle is tailored for its specific function.

For example, lysosomes have a relatively low pH of 4.5. [6] Additionally, fluorescence microscopy techniques have indicated that phagosomes also have a relatively low internal pH. [15] 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. [15]

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. [6] [16] 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. [17]

Protons being pumped from the mitochondrial matrix into the intermembrane space as the electron transport chain runs, lowering the pH of the intermembrane space. Mitochondria Intermembrane pH.jpg
Protons being pumped from the mitochondrial matrix into the intermembrane space as the electron transport chain runs, lowering the pH of the intermembrane space.

Measurement

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. [18] [19] For measuring pH inside of organelles, a technique utilizing pH-sensitive green fluorescent proteins (GFPs) may be used. [20]

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. [18] 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. [19] 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. [21]

Microelectrode

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. [19] 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. [18] [19] 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. [19]

Fluorescence spectroscopy

Another way to measure Intracellular pH (pHi) is with dyes that are sensitive to pH, and fluoresce differently at various pH values. [15] [22] This technique, which makes use of fluorescence spectroscopy, consists of adding this special dye to the cytosol of a cell. [18] [19] 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. [18] [19]

Nuclear magnetic resonance

In addition to using pH-sensitive electrodes and dyes to measure pHi, Nuclear Magnetic Resonance (NMR) spectroscopy can also be used to quantify pHi. [19] NMR, typically speaking, reveals information about the inside of a cell by placing the cell in an environment with a potent magnetic field. [18] [19] 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. [18] Additionally, NMR may also be used to reveal the presence of intracellular sodium, which can also provide information about the pHi. [23]

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. [24]

pH-sensitive GFPs

To determine the pH inside organelles, pH-sensitive GFPs are often used as part of a noninvasive and effective technique. [20] 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. [21] 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. [21] [25]

Related Research Articles

Cell biology is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living and functioning of organisms. Cell biology is the study of the structural and functional units of cells. Cell biology encompasses both prokaryotic and eukaryotic cells and has many subtopics which may include the study of cell metabolism, cell communication, cell cycle, biochemistry, and cell composition. The study of cells is performed using several microscopy techniques, cell culture, and cell fractionation. These have allowed for and are currently being used for discoveries and research pertaining to how cells function, ultimately giving insight into understanding larger organisms. Knowing the components of cells and how cells work is fundamental to all biological sciences while also being essential for research in biomedical fields such as cancer, and other diseases. Research in cell biology is interconnected to other fields such as genetics, molecular genetics, molecular biology, medical microbiology, immunology, and cytochemistry.

<span class="mw-page-title-main">Green fluorescent protein</span> Protein that exhibits bright green fluorescence when exposed to ultraviolet light

The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria and is sometimes called avGFP. However, GFPs have been found in other organisms including corals, sea anemones, zoanithids, copepods and lancelets.

<span class="mw-page-title-main">Electrophysiology</span> Study of the electrical properties of biological cells and tissues.

Electrophysiology is the branch of physiology that studies the electrical properties of biological cells and tissues. It involves measurements of voltage changes or electric current or manipulations on a wide variety of scales from single ion channel proteins to whole organs like the heart. In neuroscience, it includes measurements of the electrical activity of neurons, and, in particular, action potential activity. Recordings of large-scale electric signals from the nervous system, such as electroencephalography, may also be referred to as electrophysiological recordings. They are useful for electrodiagnosis and monitoring.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<span class="mw-page-title-main">Flow cytometry</span> Lab technique in biology and chemistry

Flow cytometry (FC) is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles.

<span class="mw-page-title-main">Immunofluorescence</span> Technique used for light microscopy

Immunofluorescence(IF) is a light microscopy-based technique that allows detection and localization of a wide variety of target biomolecules within a cell or tissue at a quantitative level. The technique utilizes the binding specificity of antibodies and antigens. The specific region an antibody recognizes on an antigen is called an epitope. Several antibodies can recognize the same epitope but differ in their binding affinity. The antibody with the higher affinity for a specific epitope will surpass antibodies with a lower affinity for the same epitope.

<span class="mw-page-title-main">Voltage clamp</span>

The voltage clamp is an experimental method used by electrophysiologists to measure the ion currents through the membranes of excitable cells, such as neurons, while holding the membrane voltage at a set level. A basic voltage clamp will iteratively measure the membrane potential, and then change the membrane potential (voltage) to a desired value by adding the necessary current. This "clamps" the cell membrane at a desired constant voltage, allowing the voltage clamp to record what currents are delivered. Because the currents applied to the cell must be equal to the current going across the cell membrane at the set voltage, the recorded currents indicate how the cell reacts to changes in membrane potential. Cell membranes of excitable cells contain many different kinds of ion channels, some of which are voltage-gated. The voltage clamp allows the membrane voltage to be manipulated independently of the ionic currents, allowing the current–voltage relationships of membrane channels to be studied.

<span class="mw-page-title-main">Patch clamp</span> Laboratory technique in electrophysiology

The patch clamp technique is a laboratory technique in electrophysiology used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is especially useful in the study of excitable cells such as neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, and can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts.

<span class="mw-page-title-main">Fluorescence microscope</span> Optical microscope that uses fluorescence and phosphorescence

A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microscope" refers to any microscope that uses fluorescence to generate an image, whether it is a simple set up like an epifluorescence microscope or a more complicated design such as a confocal microscope, which uses optical sectioning to get better resolution of the fluorescence image.

Fluorescence Loss in Photobleaching (FLIP) is a fluorescence microscopy technique used to examine movement of molecules inside cells and membranes. A cell membrane is typically labeled with a fluorescent dye to allow for observation. A specific area of this labeled section is then bleached several times using the beam of a confocal laser scanning microscope. After each imaging scan, bleaching occurs again. This occurs several times, to ensure that all accessible fluorophores are bleached since unbleached fluorophores are exchanged for bleached fluorophores, causing movement through the cell membrane. The amount of fluorescence from that region is then measured over a period of time to determine the results of the photobleaching on the cell as a whole.

Voltage-sensitive dyes, also known as potentiometric dyes, are dyes which change their spectral properties in response to voltage changes. They are able to provide linear measurements of firing activity of single neurons, large neuronal populations or activity of myocytes. Many physiological processes are accompanied by changes in cell membrane potential which can be detected with voltage sensitive dyes. Measurements may indicate the site of action potential origin, and measurements of action potential velocity and direction may be obtained.

Synapto-pHluorin is a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of a vesicle-associated membrane protein (VAMP). At the acidic pH inside transmitter vesicles, synapto-pHluorin is non-fluorescent (quenched). When vesicles get released, synapto-pHluorin is exposed to the neutral extracellular space and the presynaptic terminal becomes brightly fluorescent. Following endocytosis, vesicles become re-acidified and the cycle can start again. Chemical alkalinization of all vesicles is often used for normalization of the synapto-pHluorin signals. Synapto-pHluorin sometimes consists of yellow fluorescent protein (YFP) to monitor the cytoplasm because its pKa is higher than GFP.

<span class="mw-page-title-main">Jennifer Lippincott-Schwartz</span> American biologist

Jennifer Lippincott-Schwartz is a Senior Group Leader at Howard Hughes Medical Institute's Janelia Research Campus and a founding member of the Neuronal Cell Biology Program at Janelia. Previously, she was the Chief of the Section on Organelle Biology in the Cell Biology and Metabolism Program, in the Division of Intramural Research in the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the National Institutes of Health from 1993 to 2016. Lippincott-Schwartz received her PhD from Johns Hopkins University, and performed post-doctoral training with Richard Klausner at the NICHD, NIH in Bethesda, Maryland.

<span class="mw-page-title-main">Fluorescence in the life sciences</span> Scientific investigative technique

Fluorescence is used in the life sciences generally as a non-destructive way of tracking or analysing biological molecules. Some proteins or small molecules in cells are naturally fluorescent, which is called intrinsic fluorescence or autofluorescence. Alternatively, specific or general proteins, nucleic acids, lipids or small molecules can be "labelled" with an extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Several techniques exist to exploit additional properties of fluorophores, such as fluorescence resonance energy transfer, where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity or protein activation to be detected; another is the change in properties, such as intensity, of certain dyes depending on their environment allowing their use in structural studies.

Fluorescent chloride sensors are used for chemical analysis. The discoveries of chloride (Cl) participations in physiological processes stimulates the measurements of intracellular Cl in live cells and the development of fluorescent tools referred below.

A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.

FlAsH-EDT<sub>2</sub> Chemical compound

FlAsH-EDT2 is an organoarsenic compound with molecular formula C24H18As2O5S4. Its structure is based around a fluorescein core with two 1,3,2-dithiarsolane substituents. It is used in bioanalytical research as a fluorescent label for visualising proteins in living cells. FlAsH-EDT2 is an abbreviation for fluorescin arsenical hairpin binder-ethanedithiol, and is a pale yellow or pinkish fluorogenic solid. It has a semi-structural formula (C2H4AsS2)2-(C13H5O3)-C6H4COOH, representing the dithiarsolane substituents bound to the hydroxyxanthone core, attached to an o-substituted molecule of benzoic acid.

Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions by fluorescence properties. Two main classes of calcium indicators exist: chemical indicators and genetically encoded calcium indicators (GECI). This technique has allowed studies of calcium signalling in a wide variety of cell types. In neurons, action potential generation is always accompanied by rapid influx of Ca2+ ions. Thus, calcium imaging can be used to monitor the electrical activity in hundreds of neurons in cell culture or in living animals, which has made it possible to observe the activity of neuronal circuits during ongoing behavior.

<span class="mw-page-title-main">Intracellular transport</span> Directed movement of vesicles and substances within a cell

Intracellular transport is the movement of vesicles and substances within a cell. Intracellular transport is required for maintaining homeostasis within the cell by responding to physiological signals. Proteins synthesized in the cytosol are distributed to their respective organelles, according to their specific amino acid’s sorting sequence. Eukaryotic cells transport packets of components to particular intracellular locations by attaching them to molecular motors that haul them along microtubules and actin filaments. Since intracellular transport heavily relies on microtubules for movement, the components of the cytoskeleton play a vital role in trafficking vesicles between organelles and the plasma membrane by providing mechanical support. Through this pathway, it is possible to facilitate the movement of essential molecules such as membrane‐bounded vesicles and organelles, mRNA, and chromosomes.

<span class="mw-page-title-main">Fluorescence imaging</span> Type of non-invasive imaging technique

Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.

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