Magnetic Project

The short-term goal of this project is to replicate the finding of Xu et al that CB1 receptor is activated by the electromagnetic perceptive gene product (EPG). The long-term goal of the project is to investigate the mechanism of EPG action.

Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields.

EPG protein needs to be moved from pcDNA3 plasmid to pGHE.

HIFI Subcloning

S. XU1,2, Z. M. KRANZ3, X. ZHANG1,2, S. MITRA1,2, G. PELLED1,2,4, A. A. GILAD1,2,4;

1Biomed. Engin., 2Inst. of Quantitative Hlth. Sci. and Engin., 3Col. of Osteo. Med., 4Dept. of Radiology, Michigan State Univ., East Lansing, MI

Disclosures

S. Xu: None. Z.M. Kranz: None. X. Zhang: None. S. Mitra: None. G. Pelled: None. A.A. Gilad: None.

Abstract

Recently, we have identified and cloned the Electromagnetic Perceptive Gene (EPG) from the Kryptopterus bicirrhis (glass catfish). We have expressed the EPG in mammalian cells and demonstrated that in the presence of magnetic field there are increases of intracellular calcium (Krishnan et al., Sci Reports, 2018). This feature makes EPG a potential new technology for remote control of cellular activity. However, the mechanism by which the magnetic field activates the EPG remains to be identified. To find what proteins EPG interact with in mammalian cells, a glutathione S-transferase (GST) tag was introduced to the N-terminal of EPG which improves the protein expression and solubility in E. coli. The affinity-purification was followed by mass spectrometry (AP-MS) with the enrichment of GST tag. Pulldown was performed using extract both from rat brain and HEK293 cell. Additionally, formaldehyde crosslink immunoprecipitation with and without magnetic field was used to identify the binding partners of the EPG. Using bioinformatics, three candidates out of 300 AP-MS reads were tested: G Protein Subunit Alpha I3 (Gαi3) and O1 (Gαo), and the cannabinoid receptor 1 (CB1) which are expressed in the cerebral cortex. Moreover, the affinity between EPG and its partners appeared to be enhanced in the presence of magnetic field: the immunoprecipitation showed the CB1 pulldown by EPG under a magnetic field of 100 mT increased by 190 ±10 %. We then investigated this signal transduction pathway in mammalian cells. We used Fura-2 (1μM) to measure calcium change in induced neural progenitor cells (iNPCs) and HEK293 cells. In the presence of a magnetic field of 100 mT, 40.5±3.5% of 200 iNPCs and 25±3.0 % of 200 HEK293 cells showed Fura-2 signal increases of 2.3±0.2 and 2.0±0.2 times, respectively. However, the calcium activation was 100% abolished in 200 iNPCs and HEK293 cells after treatment of 100 ng/ml pertussis toxin which inhibits the function of CB1 through the ADP-ribosylation of the Gαi and Gαo. Furthermore, GCaMP6m and EPG were co-transfected in iNPCs. Magnetic field of 100 mT evoked 2.5±0.5 fold increase calcium responses in 36.5% of 200 cells within 10 sec. Administration of the pertussis toxin (100 ng/ml) completely abolished the stimulation, while atropine (10 μM), an antagonist of the muscarinic acetylcholine receptor that is considered unrelated to CB1 pathway, did not affect calcium responses.So far, our results suggest that CB1, Gαi3 and Gαo may interact with EPG and the interactions can be enhanced under a magnetic field. It will be important to elucidate the exact mechanism by which EPG works through, in order to optimize and improve this magnetogenetics technology.