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Calcium Probe Problems

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.


12 comments on “Calcium Probe Problems”

  1. Mad Chemist says:

    A lot of calcium in the scientific news lately. There’s also the new class of calcium dependent antibiotics discovered recently.
    A new antibiotic class would be very beneficial, if these can make it to the clinic.

  2. Fuh Dge says:

    I definitely appreciate a paper that looks at the flaws of a commonly used method and appreciate knowing the off target effects of these calcium chelating dyes. However, I think the main conclusions of the paper are pretty trite. Our lab does a lot of high-throughput screening and uses these sorts of dyes at ~1 µM at most. That’s on the lower end of dye concentration used in the cell-based assays, and 2-fold to 5-fold lower than the ex vivo/in vivo work that they do. Barring Rhod2-AM (whose effects aren’t too surprising, given its high concentration in mitochondria), the activity of the dyes at ~1 µM is negligible on the Na-K-ATPase, cell viability, glucose uptake, etc.

    I think the overall finding of the paper is something that anyone in the fields of chemical biology/pharmacology worth their salt already knew: don’t use 2-fold to 100-fold more of the probe compound than you need to. If you had a drug that’s maximally active at your target at 0.5 µM, then it’s obvious that using 50 µM of compound does nothing else for you, except wrap you up in some off target effects. I think it’s odd that people are surprised that the same thing happens with other chemical probes, like dyes.

    To comment on the GCaMP stuff at the end of the paper, our lab uses fluorescent protein-based biosensors as well. We’re a fan of them: all of the facile subcellular-targeting you can do with fusion proteins, decreased operational steps you need to run the assay, etc. are definitely advantageous. However, I think offering fluorescent protein-based biosensors as a panacea for all biosensors is dangerous. I think they should have mentioned some of the problems with fluorescent protein biosensors: wide excitation/emission spectra (which complicates multiplexing), overlapping spectra for FRET-based sensors, low extinction coefficients (ε) relative to small molecule dyes (e.g. Fluo-4, which is on the shitter end of Ca2+ sensing dyes, has an ε of 88,000 cm–1M–1, while an optimized GCaMP like GCaMP5A has an ε of 56,100 cm–1M–1) which makes detecting these biosensors in low light scenarios (e.g. on a plate reader for screening) more difficult, and the difficulty of controlling biosensor expression levels in the cells, to name a few. Moreover, I think that the finding that a >26,000 Da protein has a different set of off target activities than a dye with a MW of 1000 is pretty obvious, and to just focus on how they have differential activity on the Na-K-ATPase is misleading.

    I want to emphasize that I appreciate the paper again, however. I think the science is good. I also get annoyed with how every methods paper hypes their method as the best thing since sliced bread, so reading a paper that critically analyzes the flaws of a commonly used technique is very refreshing. However, I just think that the finding of “using too much of a dye produces off target effects” is a bit hackneyed.

    1. NJBiologist says:

      I’m glad to see this… on reading the paper, my first thoughts were 1) those sound like staggering concentrations of dye; and 2) it can’t be as simple as the authors are presenting.

    2. AM says:

      Figure 1 shows that 1 uM extracellular translates to ~100 uM inside cells.

      1. Fuh Dge says:

        Yup. To the best of my understanding, after the first figure, they use extracellular concentrations of dye throughout the paper. We use 1 µM extracellular concentration in our assays.The differences they see at those levels is not statistically significant for dyes like fluo-4.

  3. Chris Phoenix says:

    I’m curious (microscopy isn’t my field) – Have any of the sub-wavelength optical microscopy techniques made it into biology/medicine yet, in a way that’s generally useful for research?

    1. imaging guy says:

      I think you are talking about superresolution microscopy (SRM). Someone has already asked the same question on “confocal microscopy listserv” and many thought it was useful in biology. My personal view is that SRM is just a gimmick.

      1. hn says:

        Why do you think it is just a gimmick?

    2. anonymouse says:

      I briefly worked in a lab where another team got a fancy structure illumination setup. Within a couple weeks of it being set up they were imaging actin filaments in dividing lung cells. They seemed to think decent results were only were only a few experiments away (not having to fix cells in plastic for TEM images seemed to be the major benefit), but I didn’t stay long enough to hear.

  4. A chemist says:

    This is common knowledge to many communities, like ICP-MS community. It may be the 1st for the signaling community but I have read like 6 papers on this in analytical journals

  5. Barry says:

    Roger Tsien was a great scientist, more eager to find flaws in his own work than in trumpeting that he had solved more than he had. We miss him.

  6. MaJor PI ( back from the dead ) says:

    If only grad students could have done good and learned their lesson, all problems could have been solved. I had to call attorneys on my grad student she was such a dummy.

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