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Difference between revisions of "Mitochondrial membrane potential"

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{{MitoPedia
{{MitoPedia
|abbr=mtMP, Δ''ψ''
|abbr=mtMP, Δ''ι''<sub>p<sup>+</sup></sub>, Δ<sub>el</sub>''F''<sub><u>''e''</u>p<sup>+</sup></sub> [V]
|description=The '''mitochondrial membrane potential''', mtMP, is the electric part of the protonmotive [[force]], Δ''p''<sub>H+</sub>.
|description=The '''mitochondrial membrane potential''' difference, mtMP or Δ''ι''<sub>p<sup>+</sup></sub> = Δ<sub>el</sub>''F''<sub><u>''e''</u>p<sup>+</sup></sub>, is the electric part of the protonmotive [[force]], Δp = Δ<sub>m</sub>''F''<sub><u>''e''</u>H<sup>+</sup></sub>.


Δ''ψ'' = Δ''p''<sub>H+</sub> - Δ''”''<sub>H+</sub> / ''F''
:::: Δ<sub>el</sub>''F''<sub><u>''e''</u>p<sup>+</sup></sub> = Δ<sub>m</sub>''F''<sub><u>''e''</u>H<sup>+</sup></sub> - Δ<sub>d</sub>''F''<sub><u>''e''</u>H<sup>+</sup></sub>
:::: Δ''Κ''<sub>p<sup>+</sup></sub> = Δp - Δ''”''<sub>H+</sub>·(''z''<sub>H<sup>+</sup></sub>·''F'')<sup>-1</sup>


mtMP or Δ''ψ'' is the potential difference across the inner mitochondrial (mt) membrane, expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ electron or per electron moved across the transmembrane potential difference, with the number of ‘motive’ electrons expressed in the unit coulomb [C].
Δ''ι''<sub>p<sup>+</sup></sub> is the potential difference across the mitochondrial inner membrane (mtIM), expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ charge or per charge moved across the transmembrane potential difference, with the number of ‘motive’ charges expressed in the unit coulomb [C].
|info=[[Mitchell 1961 Nature]], [[Gnaiger 2020 BEC MitoPathways]]
}}
Communicated by [[Gnaiger E]] 2012-10-05, edited 2016-02-06, 2017-09-05, 2022-07-05.
:::: The chemical part of the protonmotive force, Δ<sub>d</sub>''F''<sub><u>''e''</u>H<sup>+</sup></sub> = Δ''”''<sub>H+</sub>·(''z''<sub>H<sup>+</sup></sub>·''F'')<sup>-1</sup>, stems from the difference of pH across the mt-membrane. It contains a factor that bridges the gap between the electric force [J/C] and the chemical force [J/mol]. This factor is the Faraday constant, ''F'', for conversion between electric force expressed in joules per coulomb or Volt [V=J/C] and chemical force with the unit joules per mole or Jol [Jol=J/mol],


The chemical part of the protonmotive force, ''”''<sub>H+</sub> / ''F'' stems from the difference of pH across the mt-membrane. It contains a factor that bridges the gap between the electric force [J/C] and the chemical force [J/mol]. This factor is the Faraday constant, ''F'', for conversion between electric force expressed in joules per coulomb or Volt [V=J/C] and chemical force with the unit joules per mole or Jol [Jol=J/mol],
:::::::: ''F'' = 96.4853 kJol/V = 96,485.3 C/mol


''F'' = 96.4853 kJol/V = 96,485.3 C/mol
__TOC__
|info=[[Mitchell 1961 Nature]], [[Gnaiger 2014 Preface MiP2014]]
{{Technical support integrated}}
}}
Communicated by [[Gnaiger E]] 2012-10-05, edited 2016-02-06, 2017-09-05.
{{Template:Technical support integrated}}
== Different methods for measurements of mt-membrane potential ==
== Different methods for measurements of mt-membrane potential ==


:::: mt-Membrane potential can either be measured in the [[O2k-FluoRespirometer]] fluorometrically by using the fluorophores [[TMRM]] or [[Safranin]], or potentiometrically with the [[O2k-TPP+ ISE-Module]] electrode by using the ion reporter [[TPP+]]. All mentioned ion indicator molecules inhibit respiration, which makes it essential to test the optimum concentration.
:::: mt-Membrane potential can either be measured in the [[O2k-FluoRespirometer]] fluorometrically by using the fluorophores [[TMRM]], [[Safranin]], or [[Rhodamine 123]] or potentiometrically with the [[O2k-TPP+ ISE-Module]] electrode by using the ion reporter [[TPP+]]. All mentioned ion indicator molecules inhibit respiration, which makes it essential to test the optimum concentration.


== High-resolution respirometry and mt-membrane potential ==
== High-resolution respirometry and mt-membrane potential ==


:::: The O2k-MultiSensor system provides a potentiometric and a fluorometric module for measurement of the mt-membrane potential.
:::: The O2k-MultiSensor system provides a potentiometric and a fluorometric module for measurement of the mt-membrane potential.
::::» O2k-Manual TPP: [[MiPNet15.03 O2k-MultiSensor-ISE]]
::::» [[Tetraphenylphosphonium#O2k-technical_support |TPP: O2k-technical_support]]
::::» [[MiPNet20.13 Safranin mt-membranepotential]]
::::» [[TMRM#O2k-technical_support |TMRM: O2k-technical_support]]


:::: The [[TPP+ inhibitory effect]] on respiration should be explored when applying TPP, and unspecific binding should be considered when calculating the mt-membrane potential: [[MiPNet15.08 TPP electrode]].
== Calculations ==


::::* Data analysis of [[Mitochondrial membrane potential|mitochondrial membrane potential]]  estimation using various fluorescence dyes: [[MiPNet24.09 General Template for Mt-membrane Potential Analysis]] to calculate the relative mt-membrane potential values.


=== O2k signal and output ===
::::* Data analysis of [[Mitochondrial membrane potential|mitochondrial membrane potential]] estimation with safranin using DatLab 7.4: [[MiPNet24.08 Safranin Analysis Template]] to express the mt-membrane potential values in mV.
::::# [[O2k signals and output#Signal of the O2k-Core and add-on modules |O2k signal]]: The [[O2k-TPP+ ISE-Module]] is operated through the pX channel of the O2k, with electric potential (volt [V]) as the primary and raw signal
::::# [[O2k signals and output#O2k output |O2k output]]: type C


:::: In Series D and higher the ISE connectors consist of 2 ports:
:::::* The calculation used to calculate the mt-membrane potential values are provided here complying with Oroboros transparency policy, see the following page: [[Safranin]]
::::# A BNC port: both poles are "live" so it can be used either for a combination electrode (like a classical pH) or for just one singe electrode that requires a separate reference electrode.
::::# A 2 mm pin type connection that is used for connecting the separate reference electrodes for TPP and pH.


=== Standard cleaning procedure for TPP+ and reference electrodes ===
:::::* for detailed explanation, see [[MiPNet24.11 mtMP calculation]]
::::The PVC membranes of the ISE are generally only suitable for operation in aqueous media and are damaged by non-aqueous solvents. Therefore, the necessary washing steps between experiments have to be carefully optimized according to specific experimental regimes.
== Excel analysis templates ==
:::* More advanced Excel analysis templates for the respective SUIT protocols to calculate the relative mt-membrane potential values are available on this page.
::::* The calculations used in the excel analysis template are provided complying with Oroboros transparency policy: [https://wiki.oroboros.at/index.php/Flux_/_Slope]


::::# Rinse the electrodes after use with water, dry with paper, rinse with absolute ethanol, again dry with paper and rinse with water again.
::::*''Last update 2021-09-06'': Chemical background correction was implemented into the Excel analysis templates.
::::# Store in a separate falcon tube with a proper storage solution.
::::* For [[SUIT-006 Fluo mt D034]] protocol, see template: [[File:SUIT-006 Fluo mt D034 general.xlsx]] and a demo [[File:SUIT-006 Fluo mt D034 general demo.xlsx]]
::::# When inhibitors and uncouplers have been used, continue by putting the electrodes into a falcon tube filled with distiled water for 2-5 min. Then switch to the falcon tube with a warm (30-37°C)'biological cleaning sample' for 20-30 min. Liver homogenate, isolated mitochondria or cell cultures can be used as biological cleaning sample, which can be stored at -20°C.
::::* For [[SUIT-020 Fluo mt D033]] protocol, see template: [[File:SUIT-020 Fluo mt D033 general.xlsx]] and a demo [[File:SUIT-020 Fluo mt D033 general demo.xlsx]]
::::# Alternatively, the electrodes can be washed with biological cleaning sample added to the O2k-chamber, cleaning the insterted electrodes and the O2k-chamber together.
::::* For [[SUIT-021 Fluo mt D036]] protocol, see template: [[File:SUIT-021 Fluo mt D036 general.xlsx]] and a demo [[File:SUIT-021 Fluo mt D036 general demo.xlsx]]
::::# If a carry-over of inhibitors is observed despite of the above cleaning steps, immerse the TPP electrode in absolute ethanol for a few minutes in the O2k-chamber, with stirring. However, afterwards the electrodes have to be tested and a reduced lifetime for the membranes is to be expected.
::::# [[Talk:MiPNet15.03 O2k-MultiSensor-ISE |Discussion]]


=== TPP electrode - interaction with TMPD ===
:::* Manual:[[MiPNet24.09 General Template for Mt-membrane Potential Analysis]]
::::* TMPD traveling into the TPP probe: pretty sure it was happening because the electrolyte was turning blue. And actually it became a persistent problem with several probes (at first I thought maybe there was a leak around the membrane off of one probe) (it would shift the absolute voltage values to more positive, until it the signal gets super staticky and I couldn't use the data at all). I resorted to either making sure I inverted the probe a few times to redistribute the TMPD in the electrolyte to spread it out a bit... or switching out the electrolyte (without replacing the membrane) and letting it sit overnight. And of course minimizing contact time of the probe with a solution containing TMPD.
== [[SUITbrowser]] question: Mitochondrial membrane potential ==
:::: ~ Contribution by [[Lau G|Gigi Lau]] from [[CA Vancouver Richards JG]].
:::: Several [[SUIT]] protocols are focused on the measurement of mt-membrane potential by potentiometric and fluorometric techniques.
:::: Use the [https://suitbrowser.oroboros.at/ SUITbrowser] to find the best protocol to answer this and other research questions.


=== Long-term storage of the TPP electrode ===
:::: For long-term storage (several months) disassemble the electrode, wash all parts with ethanol (sterilization), and allow all parts to dry completely. Then re-assemble the dry electrode without a membrane (avoid loosing the small parts). Store the electrode in the TPP accessory box (dark).


[[Image:O2k-Publications.jpg|left|150px|link=http://www.bioblast.at/index.php/O2k-Publications:  Topics|O2k-Publications in the MiPMap]]


=== Mitochondrial membrane potential and anoxia ===
  '''''Sort in ascending/descending order by a click on one of the small symbols in squares below'''''.
 
Default sorting: chronological. Empty fields appear first in ascending order.  
:::: Can anoxia be used as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria? This might save the time for washing out inhibitors or uncouplers. The protocol includes substrates and ADP.
{{#ask:[[Category:Publications]] [[Instrument and method::Oxygraph-2k]] [[Topic::mt-Membrane potential]]
::::# Anoxia should provide a good reference value for minimum mt-membrane potential. However, you should carry out a test experiment: After reaching anoxia, add oligomycin as a test for the possibility that ATPsynthase acts as a ATPase and thus maintains a mt-membrane potential in reversed mode of operation.  Then titrate uncoupler (FCCP) to collaps the mt-membrane potential under anoxia.
|?Was published in year=Year
::::# Careful: Ethanol as a carrier for oligomycin and FCCP exerts a chemical side effect on the TPP+ signal, which has to be evaluated in a separate control experiment and subtracted from the experimental trace.
|?Has title=Reference
 
|?Mammal and model=Organism
 
|?Tissue and cell=Tissue;cell
== Correction for substance-specific effects on the TPP signal ==
|format=broadtable
 
|limit=5000
:::: The necessity to perform a TPP chemical background experiment is explained in MiPNet 14.05. Some additional considerations:
|offset=0
 
|sort=Was published in year
=== When to apply a correction ===
|order=descending
:::: For isolated mitochondria absolute delta delta Psi values seem obtainable, see above. Approximate delta Psi values seem to be principally obtainable, though with relying on literature data. The strongly quantitative approach enabled thereby calls for complete quantifications including correction for unspecific effects.
}}
:::: For permeabilized cells, homogenates, and permeabilized fibres, absolute values of delta or delta delta Psi seem currently difficult to obtain. Data will have to presented as a relative value. Therefore, a discussion about to apply or not to apply a correction for substance specific effects seems justified: Whenever changes of mitochondrial membrane potential during an experiment are of interest a correction is most definitely needed. Otherwise even the nature of the change (increase / decrease) may be misjudged.
:::: When membrane potentials obtained by different protocols but using the same parameters (binding correction factors) during calculation should be compared to each other, correction for substance specific effects has to be done, even though only relative values are compared to each other.
:::: When relative values for membrane potentials of the same state obtained via totally identical protocols are to be compared between different samples a correction may not be strictly necessary. In this case the research will have to judge on a case basis. If the correction is obviously rather difficult,  the danger of introducing additional errors may be greater than any benefit from getting slightly more realistic values.
:::: Even if it is decided for a particular study not to apply the correction the TPP+ chemical background experiments should be done non the less to detect possible problems.
 
=== Substances ===
:::: Azide, N3-, has a very huge substance-specific effect. A correction does not seem feasible.
::::The substance-specific effect of ADP is comparably large and should be considered carefully.
 
 
== Mitochondrial membrane potential of permeabilized fibres ==
 
:::: Based on a report by [[Lin CT|Lin Chien-Te (Peter)]], [[US NC Greenville Neufer PD|Darrell Neufer at East Carolina University, Greenville, NC, USA]] and contributions by Mario Fasching and [[Sumbalova Z|Zuzana Sumbalova]].
 
=== General ===
 
:::: From experiments with isolated mitochondria or permeabilized cells one can derive the concentration and mitochondrial activity (oxygen flux per volume) necessary to obtain reliable signals with the TPP<sup>+</sup> electrode. Since unspecific binding is higher om [[Permeabilized muscle fibre|permeabilized muscle fibers]] compared to isolated mitochondria, the amount of fibres should be chosen to obtained rather high volume-specific oxygen fluxes. Even with permeabilized cells higher sample concentrations are required, as compared with standard high-resolution respiratory measurements. It is important that the total amount of TPP in the chamber is known at all times. Therefore, the sample should not be preconditioned outside of the chamber to TPP, and even a rough estimation of the sample volume will be necessary.
 
=== Introduction of the sample ===
 
:::: The established way to measure mitochondrial membrane potential for isolated mitochondria (and permeabilized cells) is to calibrate the TPP electrode by adding TPP in several steps to the experimental chamber. With the final calibration step the starting TPP<big>+</big> concentration is reached. Then the sample is injected into the "calibrated" chamber. Therefore, unlike in the application of other potentiometric methods (pH, Ca<sup>2+</sup>,..) the "calibration" does in fact serve two different purposes:
::::# Calibration of the sensor;
::::# Establishing the total amount of TPP in the chamber. This amount has to be precisely known for calculation of mtMP from the measured [TPP<sup>+</sup>].
 
:::: Introduction of the sample is a key problem. Removing the stoppers and placing the permeabilized fiber into the chamber results in a disturbance of the TPP electrode calibration. It would be necessary to replace all medium lost with medium containing exactly the TPP concentration established in the O2k-chamber before opening it. However, after introducing permeabilized fibers they immediately start to take up TPP. Opening and closing the chamber typically requires quite a lot of “bubble fighting”.
 
::::* The recommended approach is:
::::# The TPP electrode is calibrated up to 1. 0 or 1.5 ”M [TPP+] at a high O2 level of >500 nmol/ml.
::::# Lift the stopper with electrodes just slightly (stirrer off), such that the TPP electrode is still immersed in the Medium.
::::# Remove the reference electrode.
::::# Introduce fibers through reference electrode port using a glass Pasteur pipette cut to the length 16.2 cm. The cut edge was smoothed in a flame. The fibres are taken up into the pipette and gently introduced into the chamber.
::::# The wet weight is about 3 mg per chamber for mouse cardiac muscle.
::::# Close the chamber without bubbles and switch on the stirrers.
::::# Due to the high O2 flux frequent reoxygenations are required.
 
:::: The disturbance of the calibration by removing and re-inserting the reference electrode is minimal. Removal and reinsertion of the reference electrode should be done with stirrers switched off. The fibre bundles are split into several parts if necessary.
 
 
=== Reoxygenation and high oxygen ===
 
:::: The method recommended by Oroboros to do a re-oxygenation in the presence of additional electrodes is to inject H2O2 into a medium containing catalase, avoiding any mechanical disturbances, see the protocol for the MiR06 medium [[MiPNet14.13]]. Because the H2O2 method is limited to a delta cO2 of 200 ”mol/l the initial high oxygen concentration should be achieved with high oxygen in the gas pahse before starting the experiment. The O2 level can then be maintained by H2O2 injections without further opening the chamber.
 
=== Slowness of TPP uptake and release ===
:::: TPP uptake and release seems generally to be slower for permeabilzed fibers than for isolated mitochondria or permeabilized cells. However, the extent of this effect was reported to be very different by different groups. It is not yet clear what causes extremely slow uptake/ release in some cases but not in others.
 
 
== Calculation of mitochondrial membrane potential from measurement of TPP<sup>+</sup> ==
 
:::: Based on information provided in the O2k-Protocols [[MiPNet14.05 TPP-mtMembranePotential |MiPNet 14.05]], which should be consulted first. The most up to date spreadsheet templates, DatLab templates, DatLab demo files, MiPNet14.05 and its mathematical appendix can be found [[MiPNet14.05 TPP-mtMembranePotential|here]].
 
:::: The calculation of  mitochondrial membrane potential from measurements with a TPP electrode is a difficult and far from settled topic.
 
 
=== Sensitivity analysis of the method ===
[[File:Error_evaluation_absolute_1pc.png|thumb|300px|alt=absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi|absolute error in delta Psi by introduction of a 1 % error in c(TPP) plotted against delta Psi]]
 
:::: The sensitivity of the method to small errors is strongly dependent on the membrane potential. For low membrane potential the method is inherently unsuitable. This is illustrated by introducing an artificial errors in the measured [[TPP]]+ concentration and plotting the resulting errors in the calculated membrane potential against the (original) membrane potential. The exact shape of the function depends on sample amount and type, binding correction and all other external factors but the general shape is usually quite constant.
:::: Here this is illustrated for isolated (un-purified) mitochondria, simulating the effect of a +1% and -1% error in the measured TPP+ concentration.
 
:::: The used calculation template is not able to deal properly with results that would lead to a negative membrane potential, therefore errors leading to a 0 or negative membrane potential are shown here as "zero".
 
=== Unspecific binding ===
 
==== The four compartment model ====
:::: The approach to unspecific binding chosen in MiPNet 14.05 and in the Oroboros Spreadsheet temples is basically based on Rottenberg's <ref name ="Rottenberg1984">Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127-38.</ref> 4 compartment model, developed for isolated mitochondria. As shown in the mathematical appendix to [[MiPNet14.05 TPP-mtMembranePotential|MiPNet 14.05]] this approach seems to be mathematically fundamentally equivalent to the approaches by Brand <ref name="Brand1995">Brand MD (1995) Measurement of mitochondrial protonmotive force. In: Bioenergetics a practical approach (Brown GC, Cooper CE, eds):39-62. Oxford University Press, Oxford.</ref> and Kamo <ref name="Demura1987">Demura M, Kamo N, Kobatake Y (1987) Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis. Biochim Biophys Acta 903:303-8.</ref>, at least for the processes inside the mitochondria.
:::: Four compartments are considered:
 
::::# The liquid filled matrix of the mitochondria, containing “free, internal” TPP+.
::::# Material (membranes etc ) exposed to the typically high TPP+ concentration in compartment A. In Rottenbergs original approach this is the inside face of the inner mitochondrial membrane.
::::# The liquid filled space outside the mitochondria. This comprises the entire volume of the sample chamber with the exception of compartments A, B, and D.
::::# Material (membranes etc) that are exposed to the typically low TPP+ concentrations outside the mitochondrial matrix. In Rottenberg's original approach this compartment comprises the outside face of the inner mitochondrial membrane and any present material from the outer mitochondrial membrane or traces of cell material not removed during purification.
 
:::: The probe ion is supposed to accumulate in compartments B and D directly proportional to
::::* the “size/amount” of the compartment, measured by some marker,  e.g. protein content
::::* the concentration of probe molecule in the adjunct liquid phase, e.g the TPP+ concentration in the mitochondrial matrix
::::* a factor describing the affinity of the compartment to the probe molecule (the binding correction factor.
 
:::: E.g. the amount of TPP+ "bound" by the inward facing side of the inner mitochondrial membrane is
:::: ''n''(int,bound) = ''K''i' * Pmt * ''C''(int,free)
 
:::: (Equation A8a in the mathematical Appendix to MiPNet14.05)
:::: A binding correction factor (e.g. ''K''i’) is only useful together with a certain type of marker (Pmt) for which it was determined.
 
:::: The approaches by Brand and Kamo do not consider the outside compartments for unspecific binding. Indeed, for purified isolated mitochondria the outside binding seems to have a very small effect. Therefore in all further considerations one has to discern between studies of purified isolated mitochondria and studies with other sample types.
 
=== Isolated mitochondria and unspecific binding in the mitochondria ===
:::: Due to the small amount of material exposed to the outside concentrations and the low outside concentrations only the inside binding is significant.
:::: The absolute values for delta Psi will depend on the chosen binding correction factors.
 
:::: The absolute DIFFERENCE between membrane potentials (either between different states or different samples) will NOT depend on the chosen inside binding correction factor, see MiPNet 14.05 Mathematical Appendix. Therefore, it should be possible to obtain absolute change of delta Psi’s for this sample type. The insensitivity against outside binging can be shown by varying the outside binding parameter only:
 
[[File:Isolated mito Kout variation.png|500px|alt=various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta PSi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
 
:::: Only a few binding correction factors for internal binding have been published, based on rat liver mitochondria or membrane models under very different conditions (temperatures, mitochondrial membrane potential,
) While different mathematical approaches were used to describe the binding an attempt to convert these factors between different mathematical models shows quite similar values for the probe TPMP+ (the probe for which most values are available):
 
::::* Brand<ref name="Brand1995"/>: 2-3.2 ”l/mg (converted to Rottenberg's system)
::::* Rottenberg<ref name ="Rottenberg1984"/>: 2.4 to 3.7 ”l/mg
::::* Kamo<ref name="Demura1987"/>: 2.7 ”l/mg (converted to Rottenberg's system)
 
:::: Simultaneous variation of outside and inside binding parameters show:
::::* the invariance of delta delta Psi
::::* that strong deviation from published Kin' values do not lead to reasonable results:
[[File:Isolated mito KinandKout variation.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external and internal binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external = internal binding parameter Kout']]
 
:::: Conclusions for isolated isolated mitochondria:
::::* Absolute change of delta Psi values can be obtained.
::::* Precise absolute delta Psi values can not be obtained without actually measuring the binding correction factor for the studied system. Literature values will usually not be available for the desired system. Approximate delta Psi values may be obtainable by using literature values, if variances in "unspecific binding" between  sample types and conditions are small (still to be shown).
 
=== Permeabilized cells, homogenates, permeabilized fibres and unspecific binding outside the mitochondria ===
 
:::: In these sample types the determination of mitochondrial protein present is more complicated than for isolated mitochondria. Estimations may be based on the observed O2 flux, or on using a other marker for the presence of mitochondrial activity (citrate synthase). If the amount of mitochochondrial protein was estimated wrongly this may lead do drastically and obviously wrong absolute membrane membrane potentials.
:::: Below the influence of different assumption for the amount of mitochondrial protein in a preparation of brain homogenate. Delta delta Psi values between states of reasonable high membrane potential are not affected.
 
[[File:Homogenate brain centrifuged Pmt.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying amount of mitochondrial protein Pmt']]
 
:::: In these sample types there is a large amount of materials outside the mitochondrial matrix present. But potentially even more difficult than the absolute amount of material is the variety of materials. Inside the mitochondrial matrix the mitochondrial membrane is the only type of material taking up the probe ion and can therefore be accurately described by a single binding correction factor. Outside the mitochondria there may be membranes, proteins, other lipid compartments and even components of the medium to consider. It is reasonable to expect that all of them show a different affinity to TPP+ or other probe ions.
 
:::: In theory, the four compartment approach can be applied to such samples. All outside material will be exposed to the low extra-mitochondrial probe ion concentration and can therefore be included in compartment D. Due to the different nature of the outside material it can be expected that a quite different binding correction factor will be needed than the one determined by Rottenberg for the outside binding to isolated mitochondria. Additionally, it may be discussed what would be a good marker for the amount of outside material present. It should be remembered that each binding correction factor is only valid for the use with a specific marker quantity (like protein content).
:::: From a mathematical point of few the contribution of outside binding does not cancel even for the determination of change of delta Psi.
:::: However, the first question before addressing this problems is whether outside binding is relevant at all. Brand<ref name="Brand1995"/> stated that for permeabilized cells outside binding may be ignored for high mitochondrial membrane potential. Initially, this seemed to be confirmed by our own initial sensitivity studies. Using outside binding correction factors similar to the inside ones and using protein content as marker, changing the outside binding correction factor by several hundred percent caused comparable small changes in reasonable high membrane potentials and negligible changes in delta delta Psi values for permeabilized cells. However, with growing experience it became evident that unspecific binding may be underestimated by this approach, resulting in obviously too high membrane potentials  especially for states of known low potential. Part of the unreasonable high membrane potential could be explained by wrong assumptions for the amount of mitochondrial protein (Pmt). Non the less,  modeling of the outside binding correction factor showed that sometimes the correction had to be increased by factors above 25 to model reasonable membrane potential. With such a huge contribution of outside binding also differences between states (delta delta Psi) are now very significantly influenced by the choice of the outside binding correction factor. A bit surprisingly, very high membrane potentials still change only very little even when the outside binding correction factor is changed by more than a factor of 25.
 
[[File:Homogenate brain centrifuged Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'| brain homogenate, centrifuged: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
 
:::: One problem with this approach, at least in the shown example, may be that medium membrane potential values (e.g. ADP) decrease quite strongly with increasing external binding, resulting in a very strong increase in differences (changes of delta Psi), even if the low potential states are modeled not to a delta Psi of zero but one similar to the values observed for isolated mitochondria.
:::: However, in other but similar experiments medium high membrane potentials (ADP) and changes of delta Psi were more stable against variation of ''K''out'.
 
[[File:Homogenate brain crude Kout.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|crude brain homogenate: various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
 
:::: More comparative values both from isolated mitochondria and from homogenate/ permeabilized fibers of the same sample type would be necessary to evaluate this strategy.
:::: The statement that outside binding may be ignored in permeabilized cells for high membrane potentials was actually verified, only with the restriction that this hold only true for the very highest membrane potentials obtainable. This is potentially an important observation for researchers more interested in comparing just one state between different samples. It might even be argued that for very high membrane potentials an absolute delta Psi may be estimated regardless of the used binding parameters. However, this certainly needs further evaluation.
:::: By increasing both the interior and external binding parameters it is again (as with isolated mitochondria)seen that strong deviation from published Kin' values do not lead to reasonable results:
 
[[File:Homogenate brain centrifuged Koutand Kin.png|500px|alt=various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout'|various delta Psi values and one delta delta Psi values plotted against varying external binding parameter Kout']]
 
:::: There are currently no good methods known to determine the outside binding with the possible exception of radio-tracer experiments similar to those used to determine inside binding. Even if such experiments were done, due the heterogeneity and diversity of materials found in the outside compartment, the results would be less transferable to other sample types than the results for inside binding. An obvious way out would be to use a known state of zero membrane potential to determine either all or at least just the outside binding correction factor. This approach faces two problems:
::::# It is not clear how a state of reliable zero membrane potential can be reached. The true membrane potential at “low potential” states, like after addition of FCCP, may or may not be zero.
::::# As discussed above the accuracy of the entire method inherently decreases with decreasing membrane potential. At zero membrane potential the smallest error in measured TPP+ concentration will cause huge errors in delta Psi. In effect the point of lowest accuracy would be used to calibrate the entire method.
 
:::: However, at least to obtain a plausibility analysis it is certainly helpful to look at this low membrane potential states. A thorough literature search for membrane potentials obtained(with a radio-tracer method) e.g. after treatment with FCCP should be performed. Maybe an solution would be to use literature values obtained for unspecific binding in isolated mitochondria to model the inside binding but use a (crude) approximation of outside binding by observation of a zero mitochondrial membrane potential state.
 
:::: In summary, there are two obvious ways to obtain binding correction parameters that will allow a more quantitative approach:
::::* direct determination of outside binding,
::::* comparison with results obtained for isolated mitochondria under as similar as possible conditions followed by fitting the binding parameters to obtain comparable results for both types of sample preparation.
 
:::: Both approaches face several theoretical and practical differences, but should be further explored.
 
=== Further modeling options ===
:::# Saturated binding: The four compartment model could be extended by further parameters. One possibility would be to allow for a saturable component of "binding". The amount of TPP+ bound would depend only on some proportionality factor and the amount of biological material present but not on the free TPP+ concentration near the compartment. Such a behavior could be detected by performing experiments at different TPP+ levels. To obtain significant differences it would be probable necessary to use very different TPP+ concentrations (Factor 10) resulting in inhibition by TPP+ for the higher concentration studied.  This might be solved by using only results at low membrane potentials. However, at low membrane potentials the accuracy of the method is inherently low.
:::# It was suggested to use the quantity "taken up TPP+ per mass of sample (protein content)" as a relative expression for the membrane potential for given experimental conditions. One advantage is that if the result is to be a relative number anyway, it may be easier to argue (e.g. with reviewers) to use this expression than to calculate some delta Psi and than declaring: "This is not really delta Psi, but some relative value". On the downside, the comparability between different experimental conditions is certainly worse than with some calculated "relative delta Psi, plus even for the same experimental conditions the relationship between the stated number and true membrane potential (especially the linearity of the relationship!) may be worse. This should be checked by calculations / simulations.
 
:::: While probable not utilizing the measured data to its full extent this approach might be quite a safe way to present some minimum information of the data.
:::# Methods based on different kinetics of unspecific binding vs mt uptake.
 
== Simultaneous measurement of TPP and fluorescence ==
:::: A black TPP stopper is required. Light entering the chamber through the TPP electrode might be a problem. However, producing the TPP electrodes in black is presumable not a very good idea because then it will no longer be possible to "see" the position of the membrane. In preliminary tests, a chamber was equipped with TPP electrodes (black stoppers) and a fluorescence module for measuring H2O2 via the Amplex Red method. A standard TPP calibration was carried out.
:::: Amplex red / HRP addition resulted only in a minor signal in the TPP channel, a further TPP calibration showed no obvious large negative effects on the performance of the TPP electrode. A H2O2 calibration of the fluorescence signal was carried out: The '''sensitivity''' for H2O2 was reduced  by a '''factor of 3 to 4''' as compared to  measurements without TPP electrodes. At the same time the noise  was drastically increased. In the comparative experiment (without TPP electrodes) no random noise was seen - digital resolution was the limiting factor. Based on this comparison, '''noise''' increased in the experiment with TPP electrodes at least by a '''factor of 20''', probable more. It was further tested whether the presence of TPP+ alone (without electrodes) could explain this behavior but, if there were any effects at all, they were quite small. Peroxide additions corresponding to a concentration change of 110 nM (110 pmol/ml) were still well visible but not additions corresponding to a 22 nM (22 pmol/ml) concentration change. Parallel measurement e.g one chamber TPP / one chamber H2O2 are preferable.
 
:::: The O2k-system is very flexible. If you take cultured cells or isolated mitochondria, split them into the two chambers of the O2k, then you can maximize the number of simultaneously measured parameters:
::::* In both chambers oxygen concentration and oxygen flux ([[O2k-Core]]);
::::* In both chambers fluorescence, with either identical fluorophores and identical optical probes, or different ones for different parameters in each chamber (H2O2, ATP production, Ca2+, mt-membrane potential, with the potential to extend these possibilities; [[O2k-Fluo LED2-Module]]).
 
:::: Depending on fluorophore compatibility, additionally one potentiometric electrode can be inserted into each chamber (pH, TPP+, Ca2+; [[O2k-TPP%2B_ISE-Module]]);
 
:::: Or in one chamber fluorescence and the other chamber NO ([[O2k-NO_Amp-Module]]).
 
:::: Importantly, oxygen concentration is not only measured, but oxygen levels can also be controlled. This marks a new dimension in our evaluation of ROS production, with measurements spanning the entire ‘normoxic’, hyperoxic and deep hypoxic range.
 
 
== Technical service for the ISE system ==
:::: Sometimes it is difficult to find out whether problems with the ISE system are caused by the electrodes (TPP, Ca, reference, pH) or by the pX electronics of the O2k. The following tests can help to solve this question.
 
=== Testing a TPP system outside the O2k-Chamber ===
:::: This test allows to test for any disturbing influences in the chamber, like crosstalk to the [[OroboPOS]]. It is therefore similar to the test proposed in the manual (switching off the O2 polarization voltages) but has a broader scope (covering also e.g. possible interference from stirrers) and provides a more complete separation of POS an pX electrodes.
 
:::: Place TPP electrode and reference electrode together in one falcon tube filled with electrolyte solution (e.g. media). Connect the cables of the electrodes to the O2k and record the pX signal. If a problem previously observed disappears in this test (and is reproducible by using the same electrodes in the O2k-Chamber) the problem is located in the chamber, e.g. at a leaky POS membrane, see the manual. 
 
 
=== Testing a TPP system with a pH electrode (and vice versa) ===
:::: This method allows to test the sensitivity of the pX electronics.
 
::: '''Requirements'''
 
:::: Another type of electrode than can be connected to the ISE system. So if the problem is observed with an ISE system for measuring TPP, a pH electrode is the usual choice because it is available in many labs. The only requirements for the second electrode is that it can be connected via a BNC port. It does not have to be an electrode from Oroboros Instruments neither is it necessary that the electrode fits into the O2k chamber. However, note that using a pH electrode outside the O2k-Chamber also constitutes a test similar to the one described above (Testing a TPP system outside the O2k-Chamber). Therefore a problem caused e.g. by a leaky POS membrane will not be observed when testing with a pH electrode outside the chamber.
 
::: '''Procedure'''
::::* Connect the pH electrode to the oxygraph.
::::* Put the electrode into a pH 4 calibration buffer (event)
::::* Observe (record) the signal in DatLab.
::::* Put the electrode into a pH 7 calibration buffer (event). Obviously the sequence of buffers does not matter.
::::* From such a DatLab file we will see whether the pX electronics is working and even get a very rough estimation of the gain.
 
=== Testing  TPP or pH electrodes with a voltmeter ===
:::: This test measures the performance of the electrodes independently form the pX electronics of the O2k.
 
::: '''Requirements'''
 
:::: Any  voltmeter, frequently labeled "Multimeter" if different measurement modes can be selected,  suitable for measuring mV potentials. .
 
::: '''Procedure for TPP electrodes'''
::::* Set up the TPP /reference electrode in the O2k chamber as usually, only medium in the chamber.
::::* Connect the electrodes to the pX electronics and record the signal.
::::* Disconnect both electrode cables from the O2k (but leave the electrodes in the chamber, stirring still on).
::::* If you have a "Multimeter" available set in into "Voltmeter mode"
::::* Set the recording range of the voltmeter to a range good for recording about 200 mV.
::::* Measure the voltage difference between reference electrode and TPP electrode by touching (ideally fixing) the two probes of the voltmeter to the ends of the cables from the electrodes: for the reference electrodes this is simply the gold colored pin, for the TPP electrode this is the INNER , central pin. Note down the voltage reading on the voltmeter display. For this step the help of a second person is most convenient, otherwise you probable have to find  a way to fix the cables in some way. It is not necessary to have the voltmeter in contact with the electrodes all the time, just when you are reading the values.
::::* Increase the TPP concentration in a few (lets say 5) rather large steps (at least 2 ”M per step) and note down the displayed values.
::::* Finally reconnect the electrodes to the pX electronics of the O2k and observe how much (if at all) the signal has changed there.
::::* Please send us the recorded values along with the TPP concentrations used and the the DatLab file recorded at the same time (that shows the readings for the first and last point). If no change in signal is visible with the voltmeter, the problem is at the TPP electrode, not the O2k.
 
::: '''Procedure for pH electrodes'''
 
::::* Follow the procedure for TPP electrodes, moving the pH electrode between different pH calibration buffers instead of doing a TPP titration.
 
:::: '''First TPP publication with HRR''' [[Brown 2011 Am J Physiol Regul Integr Comp Physiol]]
 
== References ==
 
<references/>
 
::::» [[MiPNet15.08 TPP electrode]]
::::» [[MiPNet15.03 O2k-MultiSensor-ISE]]
::::» [[MiPNet17.05 O2k-Fluo LED2-Module]]


== Keywords: Force and membrane potential ==
{{Keywords: Force and membrane potential}}
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Revision as of 19:37, 10 July 2022


high-resolution terminology - matching measurements at high-resolution


Mitochondrial membrane potential

Description

The mitochondrial membrane potential difference, mtMP or Διp+ = ΔelFep+, is the electric part of the protonmotive force, Δp = ΔmFeH+.

ΔelFep+ = ΔmFeH+ - ΔdFeH+
Διp+ = Δp - Δ”H+·(zH+·F)-1

Διp+ is the potential difference across the mitochondrial inner membrane (mtIM), expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ charge or per charge moved across the transmembrane potential difference, with the number of ‘motive’ charges expressed in the unit coulomb [C].

Abbreviation: mtMP, Διp+, ΔelFep+ [V]

Reference: Mitchell 1961 Nature, Gnaiger 2020 BEC MitoPathways

Communicated by Gnaiger E 2012-10-05, edited 2016-02-06, 2017-09-05, 2022-07-05.
The chemical part of the protonmotive force, ΔdFeH+ = Δ”H+·(zH+·F)-1, stems from the difference of pH across the mt-membrane. It contains a factor that bridges the gap between the electric force [J/C] and the chemical force [J/mol]. This factor is the Faraday constant, F, for conversion between electric force expressed in joules per coulomb or Volt [V=J/C] and chemical force with the unit joules per mole or Jol [Jol=J/mol],
F = 96.4853 kJol/V = 96,485.3 C/mol

Template NextGen-O2k.jpg


MitoPedia O2k and high-resolution respirometry: O2k-Open Support 



Different methods for measurements of mt-membrane potential

mt-Membrane potential can either be measured in the O2k-FluoRespirometer fluorometrically by using the fluorophores TMRM, Safranin, or Rhodamine 123 or potentiometrically with the O2k-TPP+ ISE-Module electrode by using the ion reporter TPP+. All mentioned ion indicator molecules inhibit respiration, which makes it essential to test the optimum concentration.

High-resolution respirometry and mt-membrane potential

The O2k-MultiSensor system provides a potentiometric and a fluorometric module for measurement of the mt-membrane potential.
» O2k-Manual TPP: MiPNet15.03 O2k-MultiSensor-ISE
» TPP: O2k-technical_support
» MiPNet20.13 Safranin mt-membranepotential
» TMRM: O2k-technical_support

Calculations

  • The calculation used to calculate the mt-membrane potential values are provided here complying with Oroboros transparency policy, see the following page: Safranin

Excel analysis templates

  • More advanced Excel analysis templates for the respective SUIT protocols to calculate the relative mt-membrane potential values are available on this page.
  • The calculations used in the excel analysis template are provided complying with Oroboros transparency policy: [1]

SUITbrowser question: Mitochondrial membrane potential

Several SUIT protocols are focused on the measurement of mt-membrane potential by potentiometric and fluorometric techniques.
Use the SUITbrowser to find the best protocol to answer this and other research questions.


O2k-Publications in the MiPMap
Sort in ascending/descending order by a click on one of the small symbols in squares below.
Default sorting: chronological. Empty fields appear first in ascending order. 
 YearReferenceOrganismTissue;cell
Ravasz 2024 Sci Rep2024Ravasz D, Bui D, Nazarian S, Pallag G, Karnok N, Roberts J, Marzullo BP, Tennant DA, Greenwood B, Kitayev A, Hill C, KomlĂłdi T, Doerrier C, Cunatova K, Fernandez-Vizarra E, Gnaiger E, Kiebish Michael A, Raska A, Kolev K, Czumbel B, Narain NR, Seyfried TN, Chinopoulos C (2024) Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia. Sci Rep 14:1729. https://doi.org/10.1038/s41598-024-51365-4MouseHeart
Liver
Donnelly 2024 Redox Biol2024Donnelly C, KomlĂłdi T, Cecatto C, Cardoso LHD, Compagnion A-C, Matera A, Tavernari D, Campiche O, Paolicelli RC, Zanou N, Kayser B, Gnaiger E, Place N (2024) Functional hypoxia reduces mitochondrial calcium uptake. Redox Biol 71:103037. https://doi.org/10.1016/j.redox.2024.103037Human
Mouse
Heart
Skeletal muscle
Krause 2023 J Transl Med2023Krause J, Nickel A, Madsen A, Aitken-Buck HM, Stoter AMS, Schrapers J, Ojeda F, Geiger K, Kern M, Kohlhaas M, Bertero E, Hofmockel P, HĂŒbner F, Assum I, Heinig M, MĂŒller C, Hansen A, Krause T, Park DD, Just S, AĂŻssi D, Börnigen D, Lindner D, Friedrich N, Alhussini K, Bening C, Schnabel RB, Karakas M, Iacoviello L, Salomaa V, Linneberg A, Tunstall-Pedoe H, Kuulasmaa K, Kirchhof P, Blankenberg S, Christ T, Eschenhagen T, Lamberts RR, Maack C, Stenzig J, Zeller T (2023) An arrhythmogenic metabolite in atrial fibrillation. https://doi.org/10.1186/s12967-023-04420-zHumanHeart
Donnelly 2023 MitoFit2023Donnelly C, Komlódi T, Cecatto C, Cardoso LHD, Compagnion AC, Matera A, Tavernari D, Zanou N, Kayser B, Gnaiger E, Place N (2023) Functional hypoxia reduces mitochondrial calcium uptake. MitoFit Preprints 2023.2. https://doi.org/10.26124/mitofit:2023-0002 — 2024-11-17 published in Redox Biol.Human
Mouse
Skeletal muscle
Heart
Nervous system
Other cell lines
Dabrowska 2023 Int J Mol Sci2023Dabrowska A, Zajac M, Bednarczyk P, Lukasiak A (2023) Effect of quercetin on mitoBKCa channel and mitochondrial function in human bronchial epithelial cells exposed to particulate matter. Int J Mol Sci 24:638. https://doi.org/10.3390/ijms24010638HumanLung;gill
Endothelial;epithelial;mesothelial cell
Som 2023 Am J Physiol Cell Physiol2023Som R, Fink BD, Yu L, Sivitz WI (2023) Oxaloacetate regulates complex II respiration in brown fat: dependence on UCP1 expression. Am J Physiol Cell Physiol 324:C1236-48. doi: 10.1152/ajpcell.00565.2022MouseFat
Bouitbir 2022 Int J Mol Sci2022Bouitbir J, Panajatovic MV, KrĂ€henbĂŒhl S (2022) Mitochondrial toxicity associated with imatinib and sorafenib in isolated rat heart fibers and the cardiomyoblast H9c2 cell line. Int J Mol Sci 23:2282. https://doi.org/10.3390/ijms23042282RatHeart
Pallag 2022 Int J Mol Sci2022Pallag G, Nazarian S, Ravasz D, Bui D, KomlĂłdi T, Doerrier C, Gnaiger E, Seyfried TN, Chinopoulos C (2022) Proline oxidation supports mitochondrial ATP production when Complex I is inhibited. https://doi.org/10.3390/ijms23095111MouseLiver
Kidney
Tomar 2022 Biochim Biophys Acta Bioenerg2022Tomar N, Zhang X, Kandel SM, Sadri S, Yang C, Liang M, Audi SH, Cowley AW Jr, Dash RK (2022) Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla. https://doi.org/10.1016/j.bbabio.2021.148518RatHeart
Kidney
Ceja-Galicia 2022 Antioxidants (Basel)2022Ceja-Galicia ZA, GarcĂ­a-Arroyo FE, Aparicio-Trejo OE, El-Hafidi M, Gonzaga-SĂĄnchez G, LeĂłn-Contreras JC, HernĂĄndez-Pando R, Guevara-Cruz M, Tovar AR, Rojas-Morales P, Aranda-Rivera AK, SĂĄnchez-Lozada LG, Tapia E, Pedraza-Chaverri J (2022) Therapeutic effect of curcumin on 5/6Nx hypertriglyceridemia: association with the improvement of renal mitochondrial ÎČ-oxidation and lipid metabolism in kidney and liver. https://doi.org/10.3390/antiox11112195RatKidney
Fink 2022 FASEB Bioadv2022Fink BD, Rauckhorst AJ, Taylor EB, Yu L, Sivitz WI (2022) Membrane potential-dependent regulation of mitochondrial complex II by oxaloacetate in interscapular brown adipose tissue. FASEB Bioadv 4:197-210. https://doi.org/10.1096/fba.2021-00137Fat
Komlodi 2022 BEC2022Komlódi T, Tretter L (2022) The protonmotive force – not merely membrane potential. Bioenerg Commun 2022.16. https://doi.org/10.26124/bec:2022-0016
Juhaszova 2021 Function (Oxf)2021Juhaszova M, Kobrinsky E, Zorov DB, Nuss HB, Yaniv Y, Fishbein KW, de Cabo R, Montoliu L, Gabelli SB, Aon MA, Cortassa S, Sollott SJ (2021) ATP synthase K+- and H+-fluxes drive ATP synthesis and enable mitochondrial K+-"uniporter" function: I. Characterization of ion fluxes. Function (Oxf) 3(2):zqab065. doi: 10.1093/function/zqab065
Fink 2021 Pharmacol Res Perspect2021Fink BD, Yu L, Coppey L, Obrosov A, Shevalye H, Kerns RJ, Yorek MA, Sivitz WI (2021) Effect of mitoquinone on liver metabolism and steatosis in obese and diabetic rats. Pharmacol Res Perspect 9:e00701.RatLiver
Schmidt 2021 J Biol Chem2021Schmidt CA, Fisher-Wellman KH, Neufer PD (2021) From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies. J Biol Chem 207:101140. https://doi.org/10.1016/j.jbc.2021.101140
Jasz 2021 J Cell Mol Med2021JĂĄsz DK, SzilĂĄgyi ÁL, Tuboly E, BarĂĄth B, MĂĄrton AR, Varga P, Varga G, Érces D, MohĂĄcsi Á, SzabĂł A, BozĂł R, Gömöri K, Görbe A, Boros M, Hartmann P (2021) Reduction in hypoxia-reoxygenation-induced myocardial mitochondrial damage with exogenous methane. https://doi.org/10.1111/jcmm.16498RatHeart
MiPNet24.08 Safranin Analysis Template2020-05-13
O2k-Protocols
Excel template for safranin data analysis.
MiPNet25.14 TPP Analysis Template2020-##-##
O2k-Protocols
Excel template for TPP data analysis.
Aparicio-Trejo 2020 Free Radic Biol Med2020Aparicio-Trejo OE, Avila-Rojas SH, Tapia E, Rojas-Morales P, LeĂłn-Contreras JC, MartĂ­nez-Klimova E, HernĂĄndez-Pando R, SĂĄnchez-Lozada LG, Pedraza-Chaverri J (2020) Chronic impairment of mitochondrial bioenergetics and ÎČ-oxidation promotes experimental AKI-to-CKD transition induced by folic acid. Free Radic Biol Med 154:18-32.RatKidney
Gnaiger 2020 BEC MitoPathways2020Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002Human
Mouse
Heart
Skeletal muscle
Fibroblast
Lozano 2020 Part Fibre Toxicol2020Lozano O, Silva-Platas C, Chapoy-Villanueva H, PĂ©rez BE, Lees JG, Ramachandra CJA, Contreras-Torres FF, LĂĄzaro-Alfaro A, Luna-Figueroa E, Bernal-RamĂ­rez J, Gordillo-Galeano A, Benitez A, Oropeza-AlmazĂĄn Y, Castillo EC, Koh PL, Hausenloy DJ, Lim SY, GarcĂ­a-Rivas G (2020) Amorphous SiO2 nanoparticles promote cardiac dysfunction via the opening of the mitochondrial permeability transition pore in rat heart and human cardiomyocytes. Part Fibre Toxicol 17:15.RatHeart
Wang 2020 J Mol Med (Berl)2020Wang SY, Zhu Siyu, Wu Jian, Zhang Maomao, Xu Yousheng, Xu Wei, Cui Jinjin, Yu Bo, Cao Wei, Liu Jingjin (2020) Exercise enhances cardiac function by improving mitochondrial dysfunction and maintaining energy homoeostasis in the development of diabetic cardiomyopathy. J Mol Med (Berl) 98:245-61.MouseHeart
Dolezelova 2020 PLoS Biol2020DoleĆŸelovĂĄ E, KunzovĂĄ M, Dejung M, Levin M, Panicucci B, Regnault C, Janzen CJ, Barrett MP, Butter F, ZĂ­kovĂĄ A (2020) Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei. PLoS Biol 18:e3000741.Protists
Oellermann 2020 Sci Rep2020Oellermann M, Hickey AJR, Fitzgibbon QP, Smith G (2020) Thermal sensitivity links to cellular cardiac decline in three spiny lobsters. Sci Rep 10:202.CrustaceansHeart
Smith 2020 J Biol Chem2020Smith CD, Schmidt CA, Lin CT, Fisher-Wellman KH, Neufer PD (2020) Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure. J Biol Chem 295:16207-16.MouseSkeletal muscle
Cecatto 2020 Mitochondrion2020Cecatto C, Amaral AU, Wajner A, Wajner SM, Castilho RF, Wajner M (2020) Disturbance of mitochondrial functions associated with permeability transition pore opening induced by cis-5-tetradecenoic and myristic acids in liver of adolescent rats. Mitochondrion 50:1-13.RatLiver
Other cell lines
Hassan 2020 MitoFit Preprint Arch2020Hassan Hazirah, Gnaiger Erich, Zakaria Fazaine, Makpol Suzana, Abdul Karim Norwahidah (2020) Alterations in mitochondrial respiratory capacity and membrane potential: a link between mitochondrial dysregulation and autism. https://doi.org/10.26124/mitofit:200003Human
Cecatto 2020 Toxicol In Vitro2020Cecatto C, Amaral AU, Roginski AC, Castilho RF, Wajner M (2020) Impairment of mitochondrial bioenergetics and permeability transition induction caused by major long-chain fatty acids accumulating in VLCAD deficiency in skeletal muscle as potential pathomechanisms of myopathy. Toxicol In Vitro 62:104665.RatSkeletal muscle
Charles 2020 Nanomedicine (Lond)2020Charles C, Cohen-Erez I, Kazaoka B, Melnikov O, Stein DE, Sensenig R, Rapaport H, Orynbayeva Z (2020) Mitochondrial responses to organelle-specific drug delivering nanoparticles composed of polypeptide and peptide complexes. Nanomedicine (Lond) 15:2917-32.HumanEndothelial;epithelial;mesothelial cell
Malyala 2019 PLoS Comput Biol2019Malyala S, Zhang Y, Strubbe JO, Bazil JN (2019) Calcium phosphate precipitation inhibits mitochondrial energy metabolism. PLoS Comput Biol 15:e1006719.Guinea pigHeart
Fink 2019 FASEB J2019Fink BD, Yu L, Sivitz WI (2019) Modulation of complex II-energized respiration in muscle, heart, and brown adipose mitochondria by oxaloacetate and complex I electron flow. FASEB J 33:11696-705.MouseHeart
Skeletal muscle
Fat
Shuvo 2019 J Bioenerg Biomembr2019Shuvo SR, Wiens LM, Subramaniam S, Treberg JR, Court DA (2019) Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria. J Bioenerg Biomembr 51:341-54.Fungi
Spinazzi 2019 Proc Natl Acad Sci U S A2019Spinazzi M, Radaelli E, Horré K, Arranz AM, Gounko NV, Agostinis P, Maia TM, Impens F, Morais VA, Lopez-Lluch G, Serneels L, Navas P, De Strooper B (2019) PARL deficiency in mouse causes Complex III defects, coenzyme Q depletion, and Leigh-like syndrome. Proc Natl Acad Sci U S A 116:277-86.MouseNervous system
Lau 2019 Comp Biochem Physiol B Biochem Mol Biol2019Lau GY, Milsom WK, Richards JG, Pamenter ME (2019) Heart mitochondria from naked mole-rats (Heterocephalus glaber) are more coupled, but similarly susceptible to anoxia-reoxygenation stress than in laboratory mice (Mus musculus). Comp Biochem Physiol B Biochem Mol Biol 240:110375.Mouse
Other mammals
Heart
Devaux 2019 Front Physiol2019Devaux JBL, Hedges CP, Birch N, Herbert N, Renshaw GMC, Hickey AJR (2019) Acidosis maintains the function of brain mitochondria in hypoxia-tolerant triplefin fish: a strategy to survive acute hypoxic exposure? Front Physiol 9:1941.FishesNervous system
Esselun 2019 Oxid Med Cell Longev2019Esselun C, Bruns B, Hagl S, Grewal R, Eckert GP (2019) Differential effects of silibinin A on mitochondrial function in neuronal PC12 and HepG2 liver cells. Oxid Med Cell Longev 2019:1652609.Human
Rat
Nervous system
Liver
Rojas-Morales 2019 Free Radic Biol Med2019Rojas-Morales P, León-Contreras JC, Aparicio-Trejo OE, Reyes- Ocampo JG, Medina-Campos ON, Jiménez-Osorio AS, Gonzålez-Reyes S, Marquina- Castillo B, Hernåndez-Pando R, Barrera-Oviedo D, Sånchez-Lozada LG, Pedraza-Chaverri J, Tapia E (2019) Fasting reduces oxidative stress, mitochondrial dysfunction and fibrosis induced by renal ischemia-reperfusion injury. Free Radic Biol Med 135:60-67.RatKidney
Dilberger 2019 Oxid Med Cell Longev2019Dilberger B, Baumanns S, Schmitt F, Schmiedl T, Hardt M, Wenzel U, Eckert GP (2019) Mitochondrial oxidative stress impairs energy metabolism and reduces stress resistance and longevity of C. elegans. Oxid Med Cell Longev 2019:6840540.Caenorhabditis elegans
Hayward 2018 Thesis2018Hayward L (2018) The effect of anoxia on mitochondrial function in a hibernator (Ictidomys tridecemlineatus). Master's Thesis 57.Other mammalsLiver
Fisher-Wellman 2018 Cell Rep2018Fisher-Wellman KH, Davidson MT, Narowski TM, Lin CT, Koves TR, Muoio DM (2018) Mitochondrial diagnostics: A multiplexed assay platform for comprehensive assessment of mitochondrial energy fluxes. Cell Rep 24:3593-606.MouseHeart
Skeletal muscle
Menezes-Filho 2018 Biochim Biophys Acta Bioenerg2018Menezes-Filho SL, Amigo I, Luévano-Martínez LA, Kowaltowski AJ (2018) Fasting promotes functional changes in liver mitochondria. Biochim Biophys Acta Bioenerg 1860:129-35.MouseLiver
McLaughlin 2018 Biochem Biophys Res Commun2018McLaughlin KL, McClung JM, Fisher-Wellman KH (2018) Bioenergetic consequences of compromised mitochondrial DNA repair in the mouse heart. Biochem Biophys Res Commun 504:742-48.MouseHeart
Cecatto 2018 FEBS J2018Cecatto C, Amaral AU, da Silva JC, Wajner A, Schimit MOV, da Silva LHR, Wajner SM, Zanatta A, Castilho RF, Wajner M (2018) Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca2+ homeostasis in the heart. FEBS J 285:1437-55.RatHeart
Other cell lines
Komlodi 2018 J Bioenerg Biomembr2018KomlĂłdi T, Geibl FF, Sassani M, Ambrus A, Tretter L (2018) Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria. J Bioenerg Biomembr 50:355-365Guinea pigHeart
Nervous system
Smirnova 2018 Sci Rep2018Smirnova IA, Ädelroth P, Brzezinski P (2018) Extraction and liposome reconstitution of membrane proteins with their native lipids without the use of detergents. Sci Rep 8:14950.
De Carvalho 2017 Toxicol Research2017de Carvalho NR, Rodrigues NR, Macedo GE, Boligon AA, de Campos MM, Posser T, Cunha FAB, Coutinho HD, Klamt F, Bristot IJ, Merritt TJS, Franco JL (2017) Eugenia uniflora leaves essential oil promotes mitochondrial dysfunction in Drosophila melanogaster through the inhibition of oxidative phosphorylation. Toxicol Research 6:526-34 .Drosophila
Nogueira 2017 Free Radic Biol Med2017Nogueira NP, Saraiva FMS, Oliveira MP, Mendonca APM, Inacio JDF, Almeida-Amaral EE, Menna-Barreto RF, Laranja GAT, Lopes Torres EJ, Oliveira MF, Paes MC (2017) Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production. Free Radic Biol Med 108:183-91.Protists
Castellano-Gonzalez 2016 Oncotarget2016Castellano-GonzĂĄlez G, Pichaud N, Ballard JW, Bessede A, Marcal H, Guillemin GJ (2016) Epigallocatechin-3-gallate induces oxidative phosphorylation by activating cytochrome c oxidase in human cultured neurons and astrocytes. Oncotarget 7:7426-40HumanNervous system
Moon 2016 J Biol Chem2016Moon SH, Mancuso DJ, Sims HF, Liu X, Nguyen AL, Yang K, Guan S, Dilthey BG, Jenkins CM, Weinheimer CJ, Kovacs A, Abendschein D, Gross RW (2016) Cardiac myocyte-specific knock-out of calcium-independent phospholipase A2Îł (iPLA2Îł) decreases oxidized fatty acids during ischemia/reperfusion and reduces infarct size. J Biol Chem 291:19687-700.MouseHeart
Kucera 2015 Oxid Med Cell Longev2015Kucera O, Mezera V, Moravcova A, Endlicher R, Lotkova H, Drahota Z, Cervinkova Z (2015) In vitro toxicity of epigallocatechin gallate in rat liver mitochondria and hepatocytes. Oxid Med Cell Longev 2015:476180.RatLiver
Glaser 2014 Thesis2014Glaser V (2014) Efeitos da hiperglicemia cronica e seus metabolitos, metilglioxal e produtos terminais de glicacao, na fisiologia e dinamica mitochondrial no sistema nervoso central. PhD Thesis 1-117.RatNervous system
Other cell lines
Casanova 2014 Biochim Biophys Acta2014Casanova E, Baselga-Escudero L, Ribas-Latre A, Arola-Arnal A, Bladé C, Arola L, Salvadó MJ (2014) Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes. Biochim Biophys Acta 1837:783-91.RatSkeletal muscle
Other cell lines
Kukat 2014 PLoS Genet2014Kukat A, Dogan SA, Edgar D, Mourier A, Jacoby C, Maiti P, Mauer J, Becker C, Senft K, Wibom R, Kudin AP, Hultenby K, Flögel U, Rosenkranz S, Ricquier D, Kunz WS, Trifunovic A (2014) Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity. https://doi.org/10.1371/journal.pgen.1004385MouseHeart
Pham 2014 Am J Physiol2014Pham T, Loiselle D, Power A, Hickey AJ (2014) Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart. Am J Physiol 307:C499–507.RatHeart
Gnaiger 2014 MitoPathways2014
O2k-Protocols
Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp. — see 5th edition: Gnaiger 2020 BEC MitoPathways.
Human
Mouse
Heart
Skeletal muscle
Fibroblast
Sarti 2013 Int J Mol Sci2013Sarti P, Magnifico MC, Altieri F, Mastronicola D, Arese M (2013) New evidence for cross talk between melatonin and mitochondria mediated by a circadian-compatible interaction with nitric oxide. Int J Mol Sci 14:11259-76.HumanOther cell lines
Felser 2013 Toxicol Sci2013Felser A, Blum K, Lindinger PW, Bouitbir J, Kraehenbuehl S (2013) Mechanisms of hepatocellular toxicity associated with dronedarone - a comparison to amiodarone. Toxicol Sci 131:480-90.Human
Rat
Liver
Krako 2013 J Alzheimers Dis2013Krako N, Magnifico MC, Arese M, Meli G, Forte E, Lecci A, Manca A, Giuffrù A, Mastronicola D, Sarti P, Cattaneo A (2013) Characterization of mitochondrial dysfunction in the 7PA2 cell model of Alzheimer’s disease. J Alzheimers Dis 37:747-58.CHO
Duicu 2013 Can J Physiol Pharmacol2013Duicu OM, Mirica SN, Gheorgheosu DE, Privistirescu AI, Fira-Mladinescu O, Muntean DM (2013) Ageing-induced decrease in cardiac mitochondrial function in healthy rats. Can J Physiol Pharmacol 91:593-600.RatHeart
Dos Santos 2013 J Bioenerg Biomembr2013dos Santos RS, Peçanha FL, da-Silva WS (2013) Functional characterization of an uncoupling protein in goldfish white skeletal muscle. J Bioenerg Biomembr 45:243-51.FishesSkeletal muscle
Volejnikova 2013 FEMS Yeast Res2013VolejnĂ­kovĂĄ A, HlouskovĂĄ J, Sigler K, PichovĂĄ A (2013) Vital mitochondrial functions show profound changes during yeast culture ageing. FEMS Yeast Res 13:7-15.Saccharomyces cerevisiae
Fungi
Brown 2012 Am J Physiol Regul Integr Comp Physiol2012Brown JC, Chung DJ, Belgrave KR, Staples JF (2012) Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels. Am J Physiol Regul Integr Comp Physiol 302:R15-28.Other mammalsSkeletal muscle
Liver
Bustamante 2012 Alcohol2012Bustamante J, Karadayian AG, Lores-Arnaiz S, Cutrera RA (2012) Alterations of motor performance and brain cortex mitochondrial function during ethanol hangover. Alcohol 46:473-9.MouseNervous system
Kumari 2012 PLoS One2012Kumari S, Mehta SL, Li PA (2012) Glutamate induces mitochondrial dynamic imbalance and autophagy activation: preventive effects of selenium. PLoS One 7:e39382.MouseNervous system
Other cell lines
Selivanov 2012 PLoS Comput Biol2012Selivanov VA, Cascante M, Friedman M, Schumaker MF, Trucco M, Votyakova TV (2012) Multistationary and oscillatory modes of free radicals generation by the mitochondrial respiratory chain revealed by a bifurcation analysis. PLoS Comput Biol 8(9):e1002700. doi: 10.1371/journal.pcbi.1002700.RatNervous system
Albertini 2012 Aging (Albany NY)2012Albertini E, KozieƂ R, Duerr A, Neuhaus M, Jansen-Duerr P (2012) Cystathionine beta synthase modulates senescence of human endothelial cells. Aging (Albany NY) 4:664-73.HumanEndothelial;epithelial;mesothelial cell
HUVEC
Leuner 2012 Mol Neurobiol2012Leuner K, Schulz K, SchĂŒtt T, Pantel J, Prvulovic D, Rhein V, Savaskan E, Czech C, Eckert A, MĂŒller WE (2012) Peripheral mitochondrial dysfunction in Alzheimer's disease: focus on lymphocytes. Mol Neurobiol 46:194-204.HumanBlood cells
Lymphocyte
Brown 2011 Am J Physiol Regul Integr Comp Physiol2011Brown JC, Chung DJ, Belgrave KR, Staples JF (2011) Mitochondrial metabolic suppression and reactive oxygen species production in liver and skeletal muscle of hibernating thirteen-lined ground squirrels. Am J Physiol Regul Integr Comp Physiol 302:15-28.Other mammalsSkeletal muscle
Liver
Selivanov 2011 PLoS Comput Biol2011Selivanov VA, Votyakova TV, Pivtoraiko VN, Zeak J, Sukhomlin T, Trucco M, Roca J, Cascante M (2011) Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain. PLoS Comput Biol 7(3):e1001115. doi: 10.1371/journal.pcbi.1001115.RatNervous system
Chinopoulos 2011 Methods Mol Biol2011Chinopoulos C, Zhang SF, Thomas B, Ten V, Starkov AA (2011) Isolation and functional assessment of mitochondria from small amounts of mouse brain tissue. Methods Mol Biol 793:311-24.RatNervous system
Xie 2010 Acta Biochim Pol2010Xie X, Chowdhury SR, Sangle G, Shen GX (2010) Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells. Acta Biochim Pol 57:393-8.PigEndothelial;epithelial;mesothelial cell
Ziabreva 2010 Glia2010Ziabreva I, Campbell G, Rist J, Zambonin J, Rorbach J, Wydro MM, Lassmann H, Franklin RJ, Mahad D (2010) Injury and differentiation following inhibition of mitochondrial respiratory chain Complex IV in rat oligodendrocytes. Glia 58:1827-37.RatNervous system
Sommer 2010 Eur Respir J2010Sommer N, Pak O, Schörner S, Derfuss T, Krug A, Gnaiger E, Ghofrani HA, Schermuly RT, Huckstorf C, Seeger W, Grimminger F, Weissmann N (2010) Mitochondrial cytochrome redox states and respiration in acute pulmonary oxygen sensing. https://doi.org/10.1183/09031936.00013809HumanLung;gill
Endothelial;epithelial;mesothelial cell
Dikov 2010 Exp Gerontol2010Dikov D, Aulbach A, Muster B, Dröse S, Jendrach M, Bereiter-Hahn J (2010) Do UCP2 and mild uncoupling improve longevity? Exp Gerontol 45:586-95.HeLa
Fibroblast
HUVEC
Stankova 2010 Toxicol In Vitro2010Staƈková P, Kučera O, Lotková H, Rouơar T, Endlicher R, Cervinková Z (2010) The toxic effect of thioacetamide on rat liver in vitro. Toxicol In Vitro 24:2097-2103.Liver
Wrzosek 2009 Eur J Pharmacol2009Wrzosek A, Lukasiak A, Gwozdz P, Malinska D, Kozlovski VI, Szewczyk A, Chlopicki S, Dolowy K (2009) Large-conductance K+ channel opener CGS7184 as a regulator of endothelial cell function. Eur J Pharmacol 602:105-11.Pig
Rat
Heart
Endothelial;epithelial;mesothelial cell
Menna-Barreto 2009 Free Radic Biol Med2009Menna-Barreto RF, Goncalves RL, Costa EM, Silva RS, Pinto AV, Oliveira MF, de Castro SL (2009) The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction. Free Radic Biol Med 47:644-53.Protists
Soller 2007 Mol Pharmacol2007Soller M, Dröse S, Brandt U, BrĂŒne B, von Knethen A (2007) Mechanism of Thiazolidinedione-dependent cell death in Jurkat T cells. Mol Pharmacol 71:1535-44.Heart
Other cell lines
Labajova 2006 Anal Biochem2006Labajova A, Vojtiskova A, Krivakova P, Kofranek J, Drahota Z, Houstek J (2006) Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode. Anal Biochem 353:37-42.RatLiver
Schoenfeld 2004 Biochem J2004Schönfeld P, Kahlert S, Reiser G (2004) In brain mitochondria the branched-chain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition. Biochem J 383:121–28.Nervous system
Huetter 2004 Biochem J2004HĂŒtter E, Renner K, Pfister G, Stöckl P, Jansen-DĂŒrr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. https://doi.org/10.1042/BJ20040095HumanFibroblast
Pecina 2003 Biochim Biophys Acta2003Pecina P, Capkova M, Chowdhury SK, Drahota Z, Dubot A, Vojtiskova A, Hansikova H, Houstekova H, Zeman J, Godinot C, Houstek J (2003) Functional alteration of cytochrome c oxidase by SURF1 mutations in Leigh syndrome. Biochim Biophys Acta 1639:53-63.HumanEndothelial;epithelial;mesothelial cell
Fibroblast
Gregori 2002 Methods Cell Sci2002Grégori G, Denis M, LefÚvre D, Beker B (2002) A flow cytometric approach to assess phytoplankton respiration. Methods Cell Sci 24: 99-106.Plants
Eubacteria

Keywords: Force and membrane potential


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Bioblast links: Force and membrane potential - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
Fundamental relationships
» Force
» Affinity
» Flux
» Advancement
» Advancement per volume
» Stoichiometric number
mt-Membrane potential and protonmotive force
» Protonmotive force
» Mitochondrial membrane potential
» Chemical potential
» Faraday constant
» Format
» Uncoupler
O2k-Potentiometry
» O2k-Catalogue: O2k-TPP+ ISE-Module
» O2k-Manual: MiPNet15.03 O2k-MultiSensor-ISE
» TPP - O2k-Procedures: Tetraphenylphosphonium
» Specifications: MiPNet15.08 TPP electrode
» Poster
» Unspecific binding of TPP+
» TPP+ inhibitory effect
O2k-Fluorometry
» O2k-Catalogue: O2k-FluoRespirometer
» O2k-Manual: MiPNet22.11 O2k-FluoRespirometer manual
» Safranin - O2k-Procedures: MiPNet20.13 Safranin mt-membranepotential / Safranin
» TMRM - O2k-Procedures: TMRM
O2k-Publications
» O2k-Publications: mt-Membrane potential
» O2k-Publications: Coupling efficiency;uncoupling


MitoPedia concepts: Respiratory state, Recommended, Ergodynamics 


MitoPedia methods: Respirometry, Fluorometry 


MitoPedia topics: EAGLE 


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