CRY2 and TRPC1

A recent paper from theQuantumTx group of Alfredo has established a much speculated connection between cryptochrome 2 and the beneficial effects of PEMF on C2C12 myoblasts. The role of Ca²⁺ influx via TRPC1 channels and subsequent cell signaling was anticipated. The nuclear interaction between CRY2 and TRPC1 was not.

Iversen JN, Tai YK, Wu KY, Wong CJK, Lim HY, Franco-Obregón A. Magnetically Stimulated Myogenesis Recruits a CRY2-TRPC1 Photosensitive Signaling Axis. Cells. 2025 Feb 6;14(3):231. PMC free paper

The journal claims that images in their papers are public domain for educational purposes a long as proper acknowledgements are given. I’ve decided to take the hypothetical student through this publication.

The abstract and introduction

In this slide show I covered the abstract and introduction and added some images to help the reader understand where the authors were coming from.

Materials and Methods

The next second of a scientific paper is Materials and Methods. This section gives the experienced reader enough information to repeat the experiments. This section can be a source of confusion if one is not experienced in the field.

Just light baseline results … CRY2 over expression

This video has grouped together Figures 1-3. Iverson 2025 reproduced previous results showing that a magnetic field going down, with the force of gravity, is better than a magnetic field going up. This is what makes QuantumTx unique. Figure 2 demonstrates the response of C2C12 myoblasts to light. This is a baseline for when they start adding PEMF. Figure 3 presents how these cells respond to PEMF when a probable receiver, cryptochrome 2 (CRY2), is over expressed. Stop the video on bar graphs illustrating PEMF influence on cyclins B1 and D1 of the cell cycle, live cell counts, and so on.

Eliminating all magnetic fields and light results

CRY2 is a receiver of visible light and magnetic fields. Figure 4 explores how some basic growth parameters respond to elimination of visible light and ambient magnetic fields. The influence of partial light through the plastic culture dishes was examined too. (Fig 5)

Eliminating endogenous CRY2 and the source of FAD results

Some of the results of the control look very similar to the results when CRY2 is over expressed. What would happen if the C2C12 cells were altered such that they didn’t express CRY2 at all? (Fig 5) FAD is a cofactor for CRY2 and maintains its stability against degradation. Riboflavin kinase is the rate limiting enzyme for production of FAD from riboflavin. (Fig 6)

CRY2 and TRPC1 colocalize and bind to each other

Figures 8-10 provide evidence that CRY2 and the Ca²⁺ channel TRPC1colocalize and interact in the cell. I’ve added some additional thoughts based on my additional thoughts on why TRPC1 was migrating slower than expected making it appear larger than its predicted mass in an SDS PAGE gel. This is something I’ve experience in my lab days. The general thought is that proteins that have an extended structure don’t coat as well with the determent and appear slower/larger in this process. I found another example of “larger than expected” TRPC1. Ironically this was also an immunoprecipitation paper of TRPC1 with the peptidyl prolyl isomerase FKBP52. This is a chaperone protein that makes sure there are not kinks in proteins around proline residues. This interaction between CRY2 nd TRPC1 appears to be real and with properly folded TRPC1

The radical pair mechanism was given deserved discussion. Certainly reactive oxygen species could influence TRPC1 conductance, calcineurin, and gene transcription. This CRY2/TRPC1 poses a different set of questions!

31 May 2025 update

The anomalously high MW of TRPC1 can also be explained by glycosylation.

Ong HL, Chen J, Chataway T, Brereton H, Zhang L, Downs T, Tsiokas L, Barritt G. Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis. Biochem J. 2002 Jun 15;364(Pt 3):641-8. PMC free paper

This study evaluated several homemade and commercial TRP1 isoform antibodies. The authors performed a lot of immunoprecipitations. The possibility that the 92-kDa band
represents a glycosylated form of TRP-1 was tested by treating the immunoprecipitated proteins (obtained using 1.12 AP) from cell extracts with PNGase F (30 units}ml for 2 h or 18 h at 37^oC) in order to remove any N-glycosyl moieties. This treatment abolished the 92-kDa band, but yielded a new band of approx. 87 kDa. These results indicate that the 92-kDa band represents a glycosylated protein. Technology limitations of 2002 make it difficult to determine which isoforms of TRP-1 the authors were looking at.

Discussion

Iversen and coauthors had a lot to say about the radical pair mechanism and generation of reactive oxygen species from CRY2 upon exposure to PEMF and/or visible light. While I am quite fond of the rpm, I think something much more profound is going on. The model hand been PEMF increased Ca²⁺ entry throughTRPC1, binding to calcineurin, dephosphorylation NFAP, altered gene transcription… Now there is a new twist to the story that CRY2 may be entering the nucleus with TRPC1 with or without its usual htererodimer partner PER. What does this mean in terms of gene transcription? It is only my speculation that CRY2 is preferentially binding to properly folded TRPC1. At this point we do not know if the CRY2/TRPC1 binding occurs before or after it is inserted in the cell membrane. The cytoplasmic side of TRPC1 that may need refolding by FKB52 would not be exposed toCRY2 from the Golgi to the cell membrane. It is appealing to argue that properly folded, non degraded TRPC1 is a signal that can be received by the nucleus. None of this has been proven. We do know that 10 Hz 1.5mT down PEMF has a positive effect on C2C12 myoblast viability in a manner that involves FAD mound CRY2.