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Difference between revisions of "Mitochondrial membrane potential"

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{{MitoPedia
{{MitoPedia
|abbr=mt-membrane potantial
|abbr=mtMP, Δ''ψ'' [V]
}}
|description=The '''mitochondrial membrane potential''', mtMP, is the electric part of the protonmotive [[force]], Δ''p''<sub>H+</sub>.
{{MitoPedia methods}}
{{MitoPedia topics}}
This page is a guide to various wiki.oroboros.at pages that complement [http://www.oroboros.at/?O2k-multisensor-manual] with up-to-date information and discussions. The general manual for the OROBOROS Ion Selective Electrode system ([[ISE]] System) is in [http://www.oroboros.at/?O2k-multisensor [MiPNet15.03]]. While working with the potentiometric (pX) channel of the O2k, the general [[ESD|guidelines for avoiding damage to the oxygraph by ESD]] should be followed.  


A rudimentary protocol for measuring mitochondrial membrane potential (MiPNet14.05), its mathematical appendix and the most up-to-date spreadsheet templates, DatLab templates, and DatLab demo files, can be found [http://www.oroboros.at/?Protocols_tpp-membranepotential here].
Δ''ψ'' = Δ''p''<sub>H+</sub> - Δ''µ''<sub>H+</sub> / ''F''


Before measuring membrane potential via [[TPP+]] is even started, the [[TPP+ inhibitory effect]] in the studied system should be explored.How to get from the measured TPP+ concentration to the mitochondrial membrane potential: [[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]]. This covers also the influence of [[Unspecific binding]].
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].
|info=[[Mitchell 1961 Nature]], [[Gnaiger 2014 Preface MiP2014]]
}}
Communicated by [[Gnaiger E]] 2012-10-05, edited 2016-02-06, 2017-09-05.
:::: The chemical part of the protonmotive force, ''µ''<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],


Practical difficulties in applying the method to permeabilized fibers are discussed in [[Mitochondrial membrane potential of permeabilized fibers]]. Some tips for [[Cleaning the TPP+ electrodes]]Some important performance parameters of the TPP electrode are summarized on this [http://www.oroboros.at/fileadmin/user_upload/MiP2010/MiP2010_Sumbalova_poster1.pdf poster]. In any way we hope you will join us for one of our [[TPP special interest group during an International Oxygraph Course]].
:::::::: ''F'' = 96.4853 kJol/V = 96,485.3 C/mol


__TOC__
{{Technical support integrated}}
== Different methods for measurements of mt-membrane potential ==


== Mitochondrial membrane potential and anoxia ==
:::: 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.


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


Can I use anoxia as a reference state for zero (minimum) mt-membrane potential in isolated mitochondria?  I want to run experiments in series without loosing the time for washing out inhibitors or uncouplers. The protocol includes substrates and ADP.
:::: 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]]


=== Answer ===
== Calculations ==


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.
::::* 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.


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.
::::* 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.


=== See also ===
:::::* The calculation used to calculate the mt-membrane potential values are provided here complying with Oroboros transparancy policy, see the following page: [[Safranin]]
[[Mitochondrial membrane potential]]


[[ISE]]
:::::* for detailed explanaition, see [[MiPNet24.11 mtMP calculation]]


== [[SUITbrowser]] question: Mitochondrial membrane potential ==
:::: 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.


== Mitochondrial membrane potential of permeabilized fibres ==
== Keywords: Force and membrane potential ==
{{Keywords: Force and membrane potential}}


Is it possible to measure [[mitochondrial membrane potential]] of permeabilized fibres? Yes.
{{MitoPedia concepts
The examples in the Membrane Potential protocol [http://www.oroboros.at/index.php?id=protocosl_tpp-membranepotential [MiPNet14.05]] are with permeabilized cells not permeabilized fibers. In the meantime, we have also worked with isolated mitochondria - as expected this proofed to be easier than the permeabilized cells. Indeed that is the reason we started with permeabilized cells: Early experiments showed it doable but since its more difficult than isolated mitos all performance results obtained should be transferable to (or even be better with) isolated mitos. But what about [[Permeabilized muscle fibre|permeabilized muscle fibers]]?
|mitopedia concept=Respiratory state, Recommended, Ergodynamics
 
}}
 
{{MitoPedia methods
 
|mitopedia method=Respirometry, Fluorometry
=== General ===
}}
We and several of our customers are currently extending the use of the [[TPP]] electrode for measuring mitochondrial membrane potential to permeabilized fibers. Fist results show:
{{MitoPedia topics
 
|mitopedia topic=EAGLE
Yes, its possible!
}}
 
{{Labeling
Before this development started, there were basic two considerations:
|additional=MitoPedia:NextGen-O2k
 
}}
a.) we have not found any reference that describes this to have been done. So one could see it as a totally new, presumable difficult technique, that needs a lot of methods development.
 
b.) on the other hand, one can argue: what’s really the difference between permeabilized cultured cells and permeabilized fibers. Maybe a bit more of non mitochondrial cell material, but otherwise?
 
 
The first step definitely is to guess the required sample amount. From experiments with isolated mitos or permeabilized cells one can see what concentration of mitos is necessary to obtain noticeable differences in the TPP concentration. This will have to be converted (e.g. via the respiratory rate) to a amount of sample necessary for permeabilized fibers. Maybe even more sample is necessary to compensate for more "outside binding" in permeabilized fibers. Even with permeabilized cells higher sample amounts are required, as compared with standard high resolution respiratory measurements. Then there have to be some modifications in the protocol, especially how the sample is introduced. It is important that the total amount of TPP in the chamber is known at all times. It follows that the sample may not be preconditioned outside of the chamber to TPP, and even a rough estimation of the sample volume will be necessary.
If we go now totally into the realm of speculation: Might there be any specific reasons why the method just does not work with permeabilized fibers even after method development?
One could postulate that rather big junks of very hydrophobic material (fat) might "hover" up all the TPP. However, due to the far higher TPP concentration inside the mitochochondria as compared to the outside concentration (because of the membrane potential) external "unspecific binding" is usually nearly negligible for permeabilized cells......
 
 
 
=== Introduction of the Sample ===
 
 
The established was 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 indented measuring chamber. With the final calibration step the planned starting TPP concentration is reached. Then the sample is injected into the "calibrated" chamber. Therefore, unlike in the application of other potentiometric methods (pH, Ca2+,..) the "calibration" does in fact serve TWO different purposes:
1.) calibration of the sensor (of course)
2.) establishing the total amount of TPP present in the chamber. This amount has to be a precisely known for the calculation of delta Psi from the measured [TPP]
 
==== Problems ====
Introduction of the sample is a key problem in extending the TPP+ method for measuring mitochondrial membrane potential for two different reasons:
 
A.) Disturbance of the calibration itself: That is after the necessary handling steps the calibration parameters determined during the calibration run are no longer correct (due to geometry changes, ...)
 
B.) Changing the total amount of TPP preset in the chamber: If solution is lost or additional liquid has to be added during the introduction of the sample the information about the total amount of TPP+ may easily get lost:
Even for permeabilized cells, this was a major issue and was solved by injection a quite concentrated sample solution as fast as possible into the chamber: A fast injection ensured that the replacement of TPP containing medium in the chamber by the medium containing sample but no TPP+ can be treated as a simple process, not involving:
1.) any mixing of the solutions before the displacement is complete
2.) any uptake of TPP by the sample before the displacement is complete. Therefore the introduction could be treated as a simple dilution of a solution with a known TPP conc. with a solution containing no TPP. (see also Note 1)
 
All methods to introduce a sample have to consider both problems.
 
==== Possible methods ====
1.)The obvious method: '''opening the chambers''' and placing the permeabilized fiber in the chamber:
From very few and insufficient trials at Oroboros it would seem that problem I.) (loss of calibration information) is maybe less severe than initially thought or is at least less significant that problem B.). This could be checked by practicing the opening and closing the chamber with the introduction of any biological material. Typical opening and closing the camber will result in loss of liquid, necessitate the introduction of new liquid. To circumvent Problem B (change of TPP+ conc.) even in such a blind trial it would be necessary to replace all liquid lost with medium containing exactly the TPP concentration established din the chamber before opening it. However Problem B might proof crucial: After introducing permeabilized fibers they immediately start to take up TPP. Opening and closing the chamber typically requires quite a lot of “bubble fighting” and placing liquid on top of the stopper. While (pre-warmed) medium containing exactly the initial TPP+ conc. after calibration can be used for this operations, the concentration in the chamber will already be different bat this time due to TPP uptake. The problem will be the more significant the longer this operations continue and the more liquid is moved around.
 
 
2.)Introduction of the sample via '''a dedicated large additional port''': Such a port would have to be closed during operation and calibration by a plug completely filling the bore (a plug e.g. only filling the top part of the stopper would create a huge unstirred zone inside the stopper, incompatible with high resolution respirometry). The construction of such a stopper that ensures a tight fit might pose some technical problems but is probable doable. Operation could work in the following way:
* End of calibration run, top of stopper is dry
* Plug for “sample port” is removed: since top of stopper is dry no additional liquid gets into the chamber
* Sample is introduced using ?very special forceps?,  ?a biopsy needle ?,  ?a steel wire? (volume of sample introduced should be known or very small)
* To enable bubble free closing: A very small volume (just as much as necessary) volume of pre warmed medium is immediately filed into the chamber via the “sample” port and the sample port is immediately closed with the plug. A small volume will be extruded from the chamber via the injection port. Because the sample already started to take up TPP+, the concentration of the volume extruded will not be exactly known (the volume will ideally be the sample volume + the volume added before closing the port).  The volume should be kept as small as possible to minimize the error.
This method requires a special stopper and plug, the handling procedure for introducing the sample has to be very special because the diameter  of the “sample port” necessarily has to be quite small (there is just not too much space left) and introduced new problems regarding the design and closing procedure of the fitting plug. The advantage is that it is not necessary to move the electrodes.
 
3.)Using the '''existing ports''' for an approach similar to the one discussed under point two. Depending on the method found to insert the sample into the chamber, either the '''reference electrode''' or the '''TPP electrode''' would be removed from the stopper (try top). The further process would be as described above.  The closing of the bore (with the electrode) is already a quite established process. If it is possible to use the port of the reference electrode, a very small (or considering the sample volume: no) additional solution (pre-warmed containing the initial TPP concentration as a first order approximation) can be used.
Advantage: No modification of stopper required, same development of “introduction skills” necessary as for method 2. Same advantages / disadvantages in regard to “Problem B” as for Method 2
Disadvantage: possible disturbance of the calibration by moving one electrode. At least for removing and re-inserting the reference electrode this problem seems (from limited experience) to be quite small. This can be easily tested. Removal and reinsertion of electrodes should be done with stirrers switched off.
 
 
4.)Using a '''partially homogenized sample''', injecting it: probable not doable with current (and possible) permeabilization protocols.
 
5.)'''Preferred Method:''' Introduction of the sample via the '''titration port''': Requires obviously the most sophisticated tool for getting the sample in, but only minimal distortion.
 
 
==== Evaluation and solution by the Neufer Group at East Carolina University ====
 
The group of [[US NC Greenville Neufer PD|Darrell Neufer at East Carolina University, Greenville, NC, USA]], evaluated two of the discussed approaches and presented a solution. For their full contribution see the Discussion page. In summary
* the port for the reference electrode is used to introduce the sample
* the sample is split into several parts
* if necessary, the sample pieces are introduced into the chamber using a standard Hamilton syringe and the reference electrode itself
* other methods for sample insertion were tested and rejected
 
'''Until full integration of their method into this page see the contribution of the Neufer group on the [[Talk:Mitochondrial_membrane_potential_of_permeabilized_fibers|Discussion page]] for a detailed description.'''
 
 
 
 
==== Method used by the OROBOROS O2k-Team ====
The method described above was further refined by the OROBOROS O2k-Team in Innsbruck using a glass Pasteur pipette to introduce the sample into the chamber via the port for the reference electrode. For full details see the [[Talk:Mitochondrial_membrane_potential_of_permeabilized_fibers|Discussion page]].
 
 
=== Performing the measurement ===
 
==== Reoxygenation and high oxygen ===
 
The method recommended by Oroboros Instruments 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 [http://www.oroboros.at/index.php?id=protocols_miro6 MiPNet14.13]. However, if the presence of catalase in the medium is not desired or the necessary increase in oxygen concentration is larger than recommended for the MiR06 approach ( ΔcO2 ≤200 μmol/l) the method decribed by the Neufer group ion the discussion section seems to be an alternative.
 
Because the H2O2 method is limited to a delta cO2 of 200 µmol/l the initial high oxygen concentration should be achieved with the gas opahse method before the statr of the experiment. The Ow2 level can then be maintained by H2O2 injections without opening the chamber.
 
==== Slowness of TPP uptake /release ====
TPP uptake and release seems generally to be slower for permeabilzed fibers than for isolated mitochondria or permeabilized cells. However, the extend 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 the membrane potential ===
 
For issues regarding the actual calculation of the mitochondrial membrane potential, see [[Calculation of mitochondrial membrane potential from measurements with a TPP electrode]], where also the complications to the calculation introduced by the presence of non mitochondrial material is discussed.
 
A specific issue of permeabilized fibres (in contrast to isolated mitos, permeabilized cells, or homogenate) is that the sample is not distributed homogeneously. Any injections during the experiment will dilute the TPP concentration but will not dilute the sample. Therefore, all quantities depending on the amount of sample (Pcell, Pmt, Vmt(absolut)) have to be set to a fixed value (= the starting value) when using the OROBOROS calculation spreadsheets.
 
=== See also ===
[[Mitochondrial membrane potential]]
 
[[ISE]]
 
[[MiPNet14.14 PermeabilizedFibrePreparation]]
 
[[Pesta 2012 Methods Mol Biol]]

Revision as of 09:35, 21 February 2020


high-resolution terminology - matching measurements at high-resolution


Mitochondrial membrane potential

Description

The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.

Δψ = ΔpH+ - ΔµH+ / F

mtMP or Δψ 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].

Abbreviation: mtMP, Δψ [V]

Reference: Mitchell 1961 Nature, Gnaiger 2014 Preface MiP2014

Communicated by Gnaiger E 2012-10-05, edited 2016-02-06, 2017-09-05.
The chemical part of the protonmotive force, µH+ / 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

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 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.

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 transparancy policy, see the following page: Safranin

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.

Keywords: Force and membrane potential


Questions.jpg


Click to expand or collaps
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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MitoPedia:NextGen-O2k