Genetic control of intracellular pH

[Tushare]

Hey all I would like to find a way to manipulate the intracellular pH of a cell without affect all the cells in my culture. I know I can use a number of approaches to acidify of alkalinize my culture be I was hoping to find a method that would involve the transfection of something genetically encoded. This would give me modulation of intracellular pH in a small percentage of my cells in my culture. Any thoughts would be much appreciated.

 

Cheers,

 

 

[CharonY]

As a more general note (not exclusively to you but to everyone starting in bio or other sciences) it always helps to establish context when conversing with other people. For example if talking about cell cultures it would help to establish what types of cells you are talking about (bacteria, eukaryote? yeast, plant mammalian?).

That being said intracellular pH is usually relatively tightly controlled as even relatively minor shifts affect protein activity. There are specialized cells that are able to cope better with extracellular pH shifts, but in many cases this is due to superior control of intracellular pH. That being said, depending on cells there are certain ways by either disrupting homeostasis pathways and/or overexpressig certain pathways that result in overproduction of acetic or basic metabolites. Often this can result in more or less extensive cell damage, however. Again, it will depend highly on the system as certain changes will be counteracted by the cell, if they can. But if inducible cell lines have been established successfully one could seed a mixed culture, of course.

[Tushare]

I am working with dissociated neurons and I would like to figure out a way to examine a post synaptic cell during perturbation of cellular and even sub-cellular pH (at the spine heads), do you have any specific metabolic enzymes that you would suggest? Inhibition of metabolism is very destructive and very pathological, I wondering about over expression of a poly histidine or glutamic acid or aspartic acid, I'd ideally like to drop the pH from a normal 7.2-7.4 down closer to 6.8-6.5. Throwing on antimycin A has worked well in the past over an hour with my neurons but this affects both neurons at a synapse within my dish. Maybe a dominant negative of oxidative phosphorylation? I'm not sure what the effect of blocking this will be over a 24hrs period.

[CharonY]

I do not use neuronal cells, but from what I have seen that it would be tricky indeed to get a somewhat stable transfection that would yield that result (as the transfections often do not result in well-controlled quantitative changes). That being said, I presume that it may be easier trying to knock down ion channels (such as Na/H exchanger) rather than trying metabolic control (which is arguably more feasible in certain immortalized cell lines, for example). But considering that your proposed experiment is even more complex as it appears you need only to manipulate one cell at the synaptic junction I am not sure whether that is feasible either.