Fluorescent dyes and probes are wonderful things, and they have been absolutely crucial to our understanding of cellular biology. Being able to see specific protein types and cellular structures in real time through a microscope with dyes, being able to monitor things like calcium flux, oxidative stress, pH and so on through fluorescent probe molecules – there’s nothing like it. Just from a medicinal chemist’s perspective, the number of screening assays that depend on fluorescent readouts is beyond numbering.
But one of the principles of chemical biology is that you can’t just assume that modifying a protein or a test molecule is going to be silent. These aren’t circles and squares up on a board. When you tag a protein to make it fluoresce you haven’t really just put a tiny bright green asterisk next to it, and when you introduce a fluorescent probe molecule, you may have perturbed the system in ways that you haven’t expected.
That’s why I’m glad to see this new paper, even though it’s going to complicate some things. The authors (from the University of Rochester and the Children’s Research Institute) show that commonly used chemical probes to measure calcium levels are not silent actors. The probe molecules themselves turn out to be inhibitors of Na,K-ATPase, a ubiquitous and important enzyme. It’s constantly pumping sodium out (and potassium into) every mammalian cell, both of those uphill against their concentration gradients. That accounts for the ATPase activity, because it’s burning ATP to get that done – in fact, this ion hauling is such a nonstop critical function that this enzyme alone accounts for about 40% of the ATP usage in a cell. Not something to be ignored, in other words.
Specifically, the chemical probes studied were Rhod-2, Fluo-4, Fura-2, and BAPTA. A quick look through the literature finds that BAPTA and Fluo-4, especially, are still being widely used, but standard assay levels of these in various cell lines inhibit the ATPase’s activity by 30 to 80%. That causes downstream effects that no one has been taking into account. Actually, “downstream” isn’t the right word: inhibiting that ATPase directly affects some types of calcium signaling, so the problem is a fundamental one. The more complex the system you’re studying with the chemical probes, the more confounding effects there are going to be. Now, it’s long been known that such probes have their limitations, but I think it’s safe to say that no one expected one quite this direct.
Fortunately, there’s an alternative. The Tsien group introduced genetically-encoded calcium fluorescent probes (GCaMPs), small Ca-sensitive fluorescent proteins, and they don’t seem to have this problem (as you’d hope) – this paper specifically checked GCaMP3, and it doesn’t affect the Na,K-ATPase. So results from that system look more solid, while the ones with the added probe molecules, as they say, are going to have to “undergo a critical review of the data”. And there’s a lot of it. A change in calcium ion levels is one of the most basic and widely-used communication modes within the living cell, and if we’ve got some wrong ideas about it, we need to untangle things.