Patrick Ernst

Patrick Ernst has commented 1 time

2 years

Patrick Ernst

Hi, first of all thank you for highlighting this, I’m always glad when people take an interest in my work. Sorry to not address your questions sooner – I only came across this now. I hope you find these answers useful, feel free to reach out if you have any more questions. As to your questions:

1) How do you know the ChR2 is definitely at the IMM?

I know that there are some methods to isolate the IMM, but as far as I know those can be difficult. By far THE biggest piece of evidence we have that ChR2 is in the IMM is the fact that it works as intended. We know from fluorescent imaging and western blotting that ChR2-eYFP expression is colocalized with mitochondria, meaning it is either in/attached to the outer membrane (OMM), in the inter-membrane space, in the IMM, or in the mitochondrial matrix. If ChR2 were expressed in the OMM, inter-membrane space, or matrix (or improperly oriented in the IMM), activation via blue light stimulation would have little to no effect on the membrane potential of the IMM. It feels a bit cheap to just say “because it works” as an explanation, but the statistical likelihood of it working and NOT being in the IMM is low enough that it’s a satisfactory answer for me.

2) Is it possible to block or mutate the ChR2 channel to determine whether it is definitely a transfer of protons through this channel causing depolarisation?

A lot of work has been done to mutate ChR2 in order to change its physical properties (e.g. redshifting the absorption spectrum, increasing photocurrent), it has been shown that mutations can affect ion sensitivity/selectability. Cho et al were able to mutate ChR2 to significantly reduce proton conductance by a factor of ~20, so this is definitely within the realm of possibility.

However, when you take into consideration the ion-specific conductivities of ChR2 as well as comparing the intra- and extra-mitochondrial concentrations of those common ions, it follows that the majority of the depolarizing ion flow is due to protons. Schneider et al looked at ion-specific conduction of ChR2 and found that under (mostly) physiological conditions the majority (~65% by my estimate, Figure 6A) of inward current (both initial and steady-state) was due to protons, and that Ca2+ was somewhat suppressed due to competition with protons. It’s not a perfect comparison, as the ion gradients across the cell membrane will differ from those across the IMM, but it does still suggest higher conductivity of protons over other ions and that higher proton current may inhibit conductivity of other ions. Further, it is well-established that the membrane potential of the IMM is predominantly due to the strong proton gradient across the IMM – the ETC utilizes this in ATP production. It follows that taking an ion channel that preferentially conducts H+ (in circumstances where there is a low H+ gradient and large Na+ or Ca2+ gradients) and then placing it in an environment with a very large H+ gradient and much lower Na+ and Ca2+ gradients would elicit conduction almost entirely made up of protons.

Additionally, while not shown in the manuscript we did investigate possible mitochondrial Ca2+ influx (at the request of a reviewer) and found no significant change to intra-mitochondrial Ca2+, suggesting negligible Ca2+ flux through mitochondrial ChR2 activation.

3) Can you speculate as to what mechanisms induce this protection of cell viability when mitochondria are preconditioned? Has anyone looked at this?

I personally don’t know, to such an extent that it would probably be irresponsible of me to guess anything specific. The core concept is straight-forward enough – submit cells to sublethal stress, and they will adapt against that stress, making them more resilient in the near future. But past that my understanding of it is fairly limited. Luckily mitochondrial preconditioning is something that people have been using and studying for years now, typically in the context of ischemic preconditioning in neurons. Correia et al. provide a review of the topic and detail some potential mechanisms involved. Note, there may be some differences in our case, due to the difference in stressors/mechanisms between ischemic preconditioning and our photostimulation.

I recently graduated and have since left that lab/project but I know they’re currently looking into the specific mechanisms behind the cytoprotective effects we’ve seen, hopefully they find something compelling! Since publishing this paper, we have tested the effects of optogenetic mitochondrial preconditioning in the context of other more common stressors and it did have a protective effect against those as well, so the underlying mechanisms may be more similar than I think.


Y.K. Cho, D. Park, A. Yang, F. Chen, A.S. Chuong, N.C. Klapoetke, E.S. Boyden. Multimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents. J Bio Chem. 2019;294(11):P3806-3821. doi:10.1074/jbc.RA118.006996

F. Schneider, D. Gradmann, P. Hegemann. Ion selectivity and competition in channelrhodopsins. Biophys J. 2013;105(1):91-100. doi:10.1016/j.bpj.2013.05.042

S.C. Correia, C. Carvalho, S. Cardoso, R.X. Santos, M.S. Santos, C.R. Oliveira, G. Perry, X. Zhu, M.A. Smith, P.I. Moreira. Mitochondrial preconditioning: a potential neuroprotective strategy. Front Aging Neurosci. 2010;2:183. doi:10.3389/fnagi.2010.00138

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