Journal of Molecular and Cellular Cardiology
Podcast on “The Mitochondrial Permeability Transition Pore”

Listen to Elizabeth (Tish) Murphy, Christopher Baines, Andrew Halestrap, and Fabio Di Lisa as they discuss the “Mitochondria special Issue: From Basic Mitochondrial Biology to Cardiovascular Disease”. Guest Edited by Elizabeth Murphy, Don Bers and Rosario Rizzuto.

Moderator
Dr Elizabeth (Tish) Murphy, National Institutes of Health, USA (EM)

Participants
Dr Christopher Baines, University of Missouri, USA (CB)
Dr Andrew Halestrap, University of Bristol, UK (AH)
Dr Fabio Di Lisa, University of Padova, Italy (FDL)

EM:     I would like to welcome everyone to the first JMCC podcast.  We will be discussing the mitochondrial permeability transition pore, an area which has received a lot of interest recently.  Here to discuss this today are Dr Halestrap, Dr Baines and Dr Di Lisa. 

As I said, what we are going to discuss is the mitochondrial permeability transition pore and we have agreed upon a rough definition of what this is.  The mitochondrial permeability transition pore is, as most everyone knows, a pore in the inner mitochondrial membrane which allows the free passage of molecules which are less than 1.5 kilodaltons.  Opening of this pore results in the depolarisation and swelling of the mitochondria; and that the conditions that lead to the opening of the pore are debated, but it can clearly be opened under conditions of high calcium and especially when accompanied by oxidative stress, elevated phosphate concentrations and adenine nucleotide depletion.  If anyone has anything they want to add to the definition, this would probably be a good time; and to keep things in order, maybe we will do it alphabetically for the time being; Chris, do you have anything you want to add to that definition?

CB       No; looks good to me.

EM:     Dr Di Lisa?

FDL:    I would like to comment on swelling; swelling is a phenomenon that we can clearly observe in vitro, in isolated mitochondria suspended in crystalloid buffer in saline, but it is not necessarily the case in vivo when the concentration of proteins in the cytosol and in mitochondria are practically the same.  I don’t know if Andrew agrees with that?

AH:     My only comment would be that the mitochondrial protein concentration is significantly higher in the matrix.  So although the swelling will not be as great, I think the evidence from electron microscopy is in many cases the mitochondria do still swell, but I agree that it is nowhere near as great as isolated mitochondria.

CB:     I think they swell to the point of rupture certainly in the test tube, but whether that happens in vivo I think can be debated.

FDL:    I would like to add a comment that in vivo it is not necessarily the case that this swelling is a direct consequence of PTP.  There could be the starting of a process of reorganisation of the mitochondrial structure that can eventually lead to swelling.  It is very difficult to ascertain what in vivo happens, whereas it is quite clear that in vitro the opening of the pore is always followed by swelling.

EM:     I think these are very good points because the mitochondrial permeability transition pore is studied very nicely in isolated mitochondria, but how this translates to what happens in the intact cell and the techniques that one uses I think are good points.  It might be worth discussing this a little bit for a few minutes before we move on to the questions.  The question that Fabio has raised is, is swelling something that happens in vivo as a result of the MPT?  It sounds like you are a little uncertain about that, Dr Di Lisa?

FDL:    I do not think it (swelling) is a necessary consequence of PTP opening in vivo; that is my point.  For sure it is not an immediate consequence of PTP opening in many circumstances in vivo, whereas of course in isolated mitochondria in vitro, you immediately see it and in fact you are measuring the PTP through the matrix swelling.  I would also like to say that we can probably agree that the immediate consequence of the opening of the pore is mitochondrial depolarisation and this is an inevitable consequence of PTP opening.  However, as we discuss, I think later on, PTP can…

AH:     In ischemia-reperfusion it is the loss of membrane potential and hence the loss of ATP synthesis and also the breakdown of ATP that has the most serious consequence.  Swelling, which may well release cyctochrome C et cetera is probably not so important because once the ATP is gone, death will be necrotic and whether or not cyctochrome C is released is probably irrelevant.

CB:     Yes, I would agree with that, certainly under ischemia-reperfusion it is probably depolarisation and ATP loss that is the key factor.

EM:     Maybe it is worth now going through some of the questions that we thought would be of interest to the JMCC community.  One of the first ones we thought was worth discussing was what is the molecular identity of the mitochondrial permeability transition pore?  Obviously we have learnt a lot from some recent genetic studies about what it is not, but I think there is still a lot unknown about what it is.  Maybe we can start with CB - and if we can each in two minutes - summarise what we think the identity is or is not of the pore.  Chris do you want to start?

CB:     I think that the knockout data with cyclophilin D supports a role for cyclophilin D as a regulator of the pore.  It is certainly not the pore itself, from a structural point of view it is a small soluble protein so it cannot be the pore and the fact that we can overcome deletion of cyclophilin D with higher levels of calcium and other inducers, suggests that it can be bypassed, but I think the data there does support its regulatory function. 

ANT is the one that I think people have really been divided on. 

The genetic data from Wallace’s group really suggest that it is not the pore itself; it can be a regulator; it certainly seems to be important for the ability of adenine nucleotides to regulate the pore and various compounds such as atractylosideand bongkrekic acid; but it does not appear to be the pore itself.  As far as VDAC is concerned, I think that one is out of the picture now.  Certainly the work we did and also Paolo Bernardi’s Group has done with the VDAC knockout mitochondria and cells, would suggest that VDAC is not important for MPT and if anything might actually be a more of a protective protein.

That really covers the three original candidates for the mitochondrial pore.  I think cyclophilin D is definitely still around, ANT is more of a regulatory protein these days and VDAC has been thrown out.

AH:     Could I just come in on the adenine nucleotide translocase, because whilst I agree with Chris that the knockout mice show that it is not essential, I think it is important to note that the mitochondria from the knockout mice had very much lower sensitivity to calcium as well as not being sensitive to adenine nucleotides.  So one possibility is that more than one membrane protein can be acted upon by cyclophilin to produce the pore and when you remove the adenine nucleotide translocase, one of the other proteins becomes dominant.  The alternative, as Chris says, is that the ANT is regulating the critical pore component, but I do not think the data at the moment allows us to discriminate those two possibilities.

CB:     Yes, I would agree with that. 

FDL:    I am not directly involved in these kinds of studies and analysis and maybe I am bringing the topic out of the main way, but I remember a very interesting comment by Dr Halestrap about this when the work of Dr Wallace came out. I am a bit puzzled about how we can survive without adenine nucleotide translocase.  From what I know about mitochondria, it is very, very difficult to think that a heart can beat without the adenine nucleotide translocase.  Although the evidence is quite clear that the pore is still there in the absence of ANT isoforms, I still wonder how these poor mice could survive.

AH:     I can answer that, I think, because Doug Wallace knocked out ANT1 and 2 in the liver, but he did not knock out ANT 4 and the mouse mitochondrial proteome in liver shows that ANT 4 is present, so that probably there was a small residual component of ANT that was sufficient to allow the liver to survive.  But I do not think that negates the result in that the pore was no longer sensitive to adenine nucleotides so knocking out the major ANT isoforms also knocked out the major regulation by adenine nucleotides.

FDL:    I agree with you Andrew, but as you know, those mitochondria were totally insensitive to ANT inhibitors as we know them, so I am a little bit reluctant to accept that there was still some ANT around.

AH:     Those people that I have spoken to say that if there was only 5% ANT left the carboxyatractyloside titration would make it very difficult to determine that 5%, because you have been looking at the difference between 95% and 100%.

EM:     One question -- it seems that you cannot have it both ways.  If there is enough ANT there to allow the heart to survive, could that amount of ANT also participate in the pore?

AH:     If it is only 5% and it is a different isoform, there are two possible ways of looking at this.  Either the isoform ANT 4 does not form the pore, but ANT 1 does, or either ANT can form the pore, but there is so much ANT 1 or 2, whichever is appropriate, that when you remove it, the little bit of ANT 4 that is left, shows negligible pore opening in comparison to another membrane component.  And what I would suggest is that other component, or at least one of them, would be the phosphate carrier and that is certainly what our recent data suggests; and our data which is now published on that certainly suggests a role for the phosphate carrier; it binds cyclophilin D; it binds reagents which activate the pore, but there is a lot of evidence that it may too be involved and it has been known for many years that phosphate is an activator of the pore.  Of course data from Paola Bernardi’s laboratory has shown that the cyclophilin (CORRECTION IN TRANSCRIPT: should be cyclosporine D) requires the presence of phosphate in the buffer to inhibit the permeability transition pore.  It could be that both the phosphate carrier and the ANT either together, or one or the other, depending on which is prevalent, forms the pore; knock out the ANT and what is left is the phosphate carrier.  But we have not knocked out successfully, more than 80% of the phosphate carrier so far with siRNA and unfortunately with 80% of the phosphate carrier left, we still have a pore.

FDL:    As far as the phosphate carrier is concerned, the proposal by Dr Halestrap is for sure very, very interesting.  However, as we pointed out - Paolo Bernardi and I in the review on the JMCC - there is very little room for the possibility that a phosphate carrier is involved in the regulation of PTP by phosphate.  Phosphate can be both an inducer and a desensitizer of the pore and it is quite difficult that phosphate carrier participates to this regulation.  Time is going by so I do not think that there is really enough time to detail these arguments, but I think it is still possible that phosphate carrier participates to the formation of the pore.  Yet I still see very little room for participating in the regulation by phosphate.

EM:     We may want to move on; we have discussed what it is not and we have some possibilities of what it might be, but it is still very elusive in terms of what this is and this is maybe something we want to come back to in the future direction in terms of strategies for trying to identify what is the identity of the pore which will clearly make research in this area much easier to do. 

Why don’t new move on to the next question which is whether there are substates of the pore or not; whether the pore can open transiently without swelling and uncoupling?  Dr Di Lisa, do you want to start on this one?

FDL:    I think we were the first to propose this based on our measurement of calcein fluorescence and this has been confirmed directly or indirectly by many works.  I especially refer to very nice work appearing last year in the Cell by Dr Wang from a collaboration between the National Institute on Aging laboratory of Ed Lakatta and the Department of Physiology in Peking in China headed by Dr Cheng.  They demonstrated that mitochondria can produce super oxide flashes and this is through transient opening of the permeability transition pore.  This can also be a matter that we can touch upon discussing the possible physiological meaning of the permeability transition pore.  This was for sure very, very nice evidence of transient opening.  What is the role of this transient opening is very difficult to see whereas, as we know, the prolonged opening is for sure related to cell death. 

AH:     One point about the transient opening is that with our 2-deoxyglucose technique to measure poring, we always get some 2-deoxyglucose associated with the mitochondria, even in control hearts.  That might indicate transient opening; the problem is that we could never block it with cyclosporin so we never really felt we could conclude it, but we were seeing transient opening.  What we do know is that we can reverse the pore opening once it has opened, so on reperfusion some pores that were opened close again, so in that sense we know it is reversible in vivo.

But, I think the other point that we are uncertain about is whether there is a substate in the conductance of the pore as some have suggested from electric physiological measurements and I would be interested in other people’s opinion on that because my own feeling is transient pore openings still is the full pore.  It is just opened for a short time, but others may want to suggest an alternative.

CB:     Certainly the way I read that, that is the definition I would apply, a transient short-lived, but full blown opening of the pore as opposed to the prolonged opening we see with pathology.  This is nothing we have looked at ourselves, but if we are say suggesting, for example, cyclophilin D, ANT and the phosphate carrier, I would have to wonder that under physiological conditions, presumably these guys are just fulfilling their “official function” as translocators, chaperones, whatever and would the MPTP even exist under physiological conditions and that maybe the 2-deoxyglucose and the calcein are measuring something else, another ion channel; but like I said this is nothing I have looked at personally so I am still open to suggestions.

AH:     One possibility we thought of was actually the adenine nucleotide translocase every now and then might find itself without an ATP on it and at that point it flips into the pore and then ATP binds again and it flips out.  Because we know oxidative stress is very good at preventing ATP binding to the adenine nucleotide translocase, you go from being transient, then just occasionally the pore gets to that conformation, to much more permanently when the adenine nucleotides are not binding properly.

EM:     Aren’t you suggesting that the ANT is part of the pore?

AH:     That is what I am saying; I still think it is a possibility and of course if you have two membrane components that can - depending on the conditions - form the pore, be it the phosphate carrier and the ANT, both of which bind cyclophilin, then if something affects the ANT that is the pore you will see.

EM:     Since we are discussing the transient opening that leads us into the next issue we were going to discuss which is, does the pore exist under physiological conditions and if so what might be its function?  Dr Halestrap, do you want to start on that perhaps?

AH:     In terms of it existing, I think it certainly does not open readily in normal mitochondria, otherwise we would not have oxidative phosphorylation; but there is something to be said for the idea that individual mitochondria may undergo permeability transition and perhaps as John Lemasters has suggested, as a way of removing them through autophagy or mitophagy as he calls it, so when an individual mitochondria has gone through a lot of oxidative stress which means its genome is probably going to be corrupted, then it is also likely to undergo the permeability transition and this might be a recognition mechanism whereby those sick mitochondria are taken out of the cell by autophagy.  That would be my best bet; the problem is that the cyclophilin D knockout mice seem to show very little phenotype that would support that.

FDL:    Yes, but please remember as you know this, cyclophilin D knockout still have the pore and the cyclophilin D is not the pore, so also the argument that in the absence of cyclophilin D there is apoptosis is not against the involvement of PTP in apoptosis in my opinion. 

As far as the physiology is concerned, if I can add some further comments, we have plenty of evidence that individual mitochondria can undergo depolarisation continuously in living myocytes.  If this depends on PTP opening, we still do not know.  For instance Brian O’Rourke is somewhat against this, but Wang’s paper is strongly supportive of this possibility.  Why should it be that way?  There could be many functions for this transient and repeated opening of the pore; the pore can act as a fast calcium release channel.  It can also be a way to discharge pyridine nucleotides that are mostly compartmentalised within mitochondria and in this way pyridine nucleotides could become available for increased demand by cytosol at the level of steroidogenesis for instance in mitochondria in adrenal glands or for DNA repair by the activation of the poly ADP ribose polymerase, but at this point, these are just hypotheses or conjectures and there is nothing really proven.

CB:     We could potentially add to that list, although it goes back to the whole swelling question; maybe transient opening is a way of transiently altering the volume of the mitochondria which obviously has effects on metabolic functions.  It all fits in with what we know from a pathological point of view, but under a more limited situation under physiology.  As Dr Di Lisa says, it is all up in the air right now and to be honest, until we find out what is the pore, it will be hard to get a handle on what it is doing physiologically.

EM:     To summarise what I am hearing, there are two possibilities: one is that there is some physiological role potentially for the pore, particularly maybe in a transient or sub-conductive state.  We talked a little bit a few minutes ago about it may not fully open and obviously if it had sub-conductive states that may have the physiological role, some of which Dr Di Lisa mentioned.

The other possibility that Dr Halestrap spoke of is that it maybe that under some circumstances the proteins, for example the ANT misfunctions almost and acts as a transient pore, but that would not have a physiological function.  Does that summarise some of the possibilities, or does anyone have any comments on that summary?

AH:     I think we want to make sure we added the autopagy or mitopagy of mitochondria as a real possibility.

EM:     As a way to remove damaged mitochondria?

AH:     Yes, because otherwise the mitochondria with damaged DNA will reproduce at the expense of the other ones and you will end up with a mitochondrial disease state.

EM:     Again we might want to come back to this in our future directions, but at this point maybe we should move on to another issue which is: what is the role of the pore in cardioprotection?  Obviously there has been a lot of data suggesting that it has a role; Chris, do you want to start on this?

CB:     I think the first evidence came from labs like Dr Halestrap’s and Dr Di Lisa’s showing that cyclosporin A, which inhibits cyclophilin D, does protect against the ischemia-reperfusion and again both those labs have shown that the MPTP does open upon reperfusion in particular.  Cyclophilin D knockout mice are protected against ischemia-reperfusion so certainly the MPTP plays a role in the progression of myocyte death in this particular disease model.  Obviously the question that builds upon this are cardioprotective interventions, such as preconditioning, post conditioning; however you want to do it.  Do they impinge upon the MPTP and I think that data is now beginning to amass that these particular cardioprotective stimuli do at least terminate at least partially on inhibition of the MPTP and that is why they are protective.  I would say, yes, the inhibition of MPTP is indeed cardioprotective.

AH:     I think the key thing is if the pore opens in a significant number of mitochondria, there is no doubt that the cell is going to suffer from ATP deprivation and in the heart that will ultimately lead to necrosis.  If you can block the pore with cyclosporin or some of these other treatments there is no doubt you will get cardioprotection; but the question I think is how some of these protective regimes - and particularly preconditioning - block the pore.  I do not think there is now much doubt, as Chris says that the pore is blocked.  The question is how and I have to say that that is still something of a mystery.

FDL:    I think that there are three different matters in this topic and the three of them are very difficult to address.  The very first is how we reach the pore through preconditioning and post conditioning; the second one is if we need PTP opening also for protecting the heart , not only for damaging it; and the third one, although we are sure (there is a consensus) that inhibiting the pore results in cardioprotection, probably a philosophical question could be what are we protecting from, or what is downstream of pore opening. This is especially hard to address in the heart.  We know that if we inhibit completely the respiratory chain with cyanide, or we add FCCP to heart, we obtain a sort of protection from the explosive damage that occurs upon reperfusion. The heart of the myocyte dies later, but not immediately upon reperfusion.

I still wonder why the opening of the pore is so deleterious for the heart and is not resembling simply the depolarisation of mitochondria; there has to  be something else.  For instance I think that the redistribution of NAD leading to NAD depletion could be something very important because this will arrest completely oxidative metabolism, but there could be also something else linking the opening of the pore with the rupture of sarcolemma in a matter of seconds.

AH:     I think that the other thing that is important to note is that when the pore opens it does appear that you get an additional burst of reactive oxygen species; you actually get a progression of depolarisation, ATP starts breaking down, but also increase in reactive oxygen species and that then is going to have additional effects by modifying lipids, so now phospholipases are more active; you have got the elevated calcium because you can’t make ATP to pump it out.  There is a whole load of things coming together when the pore opens which I think is much more damaging than just having an uncoupler or respiratory chain inhibitor.  The interesting thing of course is the way the infarct progresses and the idea that you have permeability transition ROS production which produces a ROS induced ROS production is a very interesting one for the spreading wave of the infarct.

FDL:    I totally agree with you Andrew. ROS are for sure is a big thing downstream of the opening of the pore; the thing is complicated by the fact that this is a vicious cycle because ROS sensitise the PTP to calcium and at the same time the opening of PTP favours ROS formation; but eventually this is very, very deleterious for the heart and if we had this together with NAD, ATP depletion and so on, then it is no wonder that the cell cannot survive.

CB:     What I was going to add was particularly with regards to the rupture of the sarcolemma question; we all tend to draw mitochondria just sort of floating in a cell, but obviously in the heart there is a lot of mitochondria just under the sarcolemma and if as you have already said, we get this sudden burst in ROS due to pore opening and micro domain of huge amounts of calcium released from the mitochondria right next to the sarcolemma, both ROS and calcium do horrible things to phospholipids so just this concentration of these toxic metabolites right next to the sarcolemma may directly contribute to rupture ultimately.

EM:     What are your thoughts on Derek Yellon’s study showing that cyclosporin would block preconditioning so that blocking transient opening prior to the sustained ischemia blocks protection?  How does that fit into the model?

AH       My problem is that we never saw any pore opening with our deoxyglucose technique during the preconditioning stimulus and we never got any evidence for pore opening; and when we isolate mitochondria after the preconditioning stimulus we really do not see any evidence for that activation of pore opening.  I don’t know how to explain their data, but certainly our own data does not support transient pore opening as a preconditioning stimulus.

CB:     I can't remember, did they show that FK506 was without effect when they did the preconditioning stimulus, because obviously
cyclosporin A also inhibits calcineurin and you may just be hyper activating a kinase or something else, so that could also explain why there is no protection at that point.

AH:     I think they may have used either NIM 811 or Debio-025 but I can’t remember.

EM:     One issue that we have discussed and that was suggested as something we might discuss is methods for measuring MPT opening, particularly in an intact heart, is it worth spending a few minutes discussing the reliability of these methods?

AH:     It is a real problem in the whole heart; obviously in the isolated heart cells, the various confocal fluorescent techniques work quite well although you have to be so careful that the very process of measuring pore opening with fluorescence does not itself activate pore opening.  In the whole heart, the deoxyglucose technique which we introduced is a very tedious technique.  I think it has a lot of merits, but it is not a perfect technique.  FDL’s technique with NAD is indirect, but it gives an indication.  It is not an easy measurement to make and it would be lovely if somebody could think of something better.

EM:     Fabio, do you have anything to add?

FDL:    As I wrote in another review I am still fascinated by the fact that two different and independent techniques give exactly the same evidence that PTP opens during reperfusion after prolonged ischemia.  As far as the intact heart is concerned, we have nothing better than what I and Andrew developed, whereas I think the calcein technique is still very valid for studying the PTP in intact cells.

EM:     One of the points that might be worth making is that many people use just membrane depolarisation in many papers which obviously is not as rigorous as the techniques that you have discussed.  I think it tells us something, but I am not sure that one can take membrane depolarisation as an indication of pore opening?  What are your thoughts on that?

CB:     I do not think so; it could be indicative, but not necessarily.  It can change without the pore opening; I think it is maybe useful as a starting point, but then ultimately you have to use one of the other techniques that have already been discussed to really hammer home if it is MPTP.

AH:     If you can add to the depolarisation that the depolarisation is inhibited by a cyclosporin analogue that has no effects on calcineurin that is better evidence, but it is still not perfect.

FDL:    Yes and I would like to put a word of caution also on the use of hydrogen peroxide or flashlights to induce the pore and just measure mitochondrial depolarisation.  This is not necessarily opening of the pore and to translate those effects and those results to the in vivo heart is really unreliable I would say.  I would also like to express a word of caution for all the studies that obtain the opening of the pore in mitochondria isolated from a given heart preparation.  This does not mean that the pore was opening in situ; it just gives you an idea that the mitochondria are less stable, but this does not say anything about the control of the pore, the moment when the pore opens and the effect of pore opening in situ.

AH:     Ideally you want to do both the in vivo or in situ measurements and then also look at the regulation of the isolated mitochondria to give you some indication of what is controlling the pore opening under those conditions.  For example with preconditioning the deoxyglucose will show less pore opening in the intact heart.  The isolated mitochondria, when you have them isolated also are less sensitive to calcium and our own studies suggest that is because they have less oxidative stress; so you are beginning to probe a little of the mechanism if you do the in vitro work.

FDL:    You were right because you always perform the two things, whereas relying just on the isolated mitochondria can give really very unreliable conclusions.

EM:     We are coming to the end of our discussion and obviously we have identified a lot that we don’t know and I guess the question that I would like to ask each of you is what are the areas for future?  What do we need to learn?  Where should we put our effort?  Fabio, do you want to start on this?

FDL:    Thank you so much.  For some reason and good reasons the study of the relationship between PTP and the heart was focussed on ischemia-reperfusion, but now we have very good evidence that we can expand the importance of the pore beyond ischemia-reperfusion, the work by Jeff Molkentin appeared in October 2007 in JCI were very nice evidence of that.  He induced calcium overload by over expressing a part of the calcium channel and he obtained hypertrophy and this phenotype was totally rescued by cyclophilin D knockout.  Cyclophilin D knockout in the same paper was shown to also abrogate the deleterious effects of doxorubicin and there is also evidence that PTP inhibition is good for the maintenance of endothelial viability, although there are no studies that I know showing that the inhibition of the pore either pharmacologically or genetically is somewhat efficacious against the atherosclerosis we see in vivo.  I think that the future will give us a lot of nice evidence of the importance of the pore in many cardiac diseases.

CB:     Yes, I would add to that certainly work from Dr Di Lisa and Paolo Bernardi’s Group and also Jeff Molkentin have been important.  I’m also looking at muscular dystrophy which we all think is just skeletal muscle, where actually one of the major problems is the heart disease that is associated with that.  Whether you inhibit cyclophilin D pharmacologically or genetically, that really seems to delay the development of cardiac failure in various models of muscular dystrophy.  That is yet another model where this MPTP seems to play a critical role in disease progression and could therefore be a target for therapeutic intervention.

AH:     To me the key areas are establishing the molecular identity.  We all would agree and I think there is a lot more needs to be done there with ultimately reconstitution and knockout studies giving confirmation.  The other area I think I am more and more convinced by is that the key to opening the pore and the key to protection is actually matrix ROS production and what regulates matrix ROS production and how things like preconditioning affect that.  Certainly our own experiments would suggest that if we could understand how ROS production was regulated, we would have a lot of scope for intervention.

EM:     Why do you think it has been so difficult to identify the components of the MPT?

AH:     Working with membrane proteins is difficult - that is one reason; and I think if you could just isolate individual proteins from the membrane in an active form, we would be there by now, but reconstituting mitochondrial membrane proteins is extremely difficult.

CB:     A lot of them are very sticky so you do a pull down and they all want to come too and the question is teasing out who is just along for the ride and who is actually involved in the process.

FDL:    May I add that although we know at this point the molecular structure of several channels and Roderick MacKinnon got the Nobel Prize for that – there is no defined structure for mitochondrial channels, not a single one.  Also people like us that are involved in calcium homeostasis of mitochondria were puzzled by the identification of calcium uniporter that later on was no longer tenable and so we do not know anything about mitochondrial channels in terms of molecular identity not only of the PTP.

CB:     I think one thing we may ultimately have to deal with is that although I would like to think that at its core the MPTP is conserved throughout different tissues – in fact we may be dealing with differences particularly from a regulatory point of view.  At least in our hands, in liver mitochondria (MPT) will pop open a heck of a lot easier than in heart mitochondria - which make sense physiologically - but that must mean there must be some differences, from a molecular make up point of view, that we may ultimately still have to deal with.