Engineered Red Opto-mGluR6 Variants as Red-shifted Optogenetics Tools to Induce Action Potential in HEK-GIRK Cells

Hoda Shams Najafabadi1 , Zahra-Soheila Soheili1 *, Mehdi Sadeghi2 , Hamid Ahmadieh3 , Hamid Gholami Pourbadie4 , Amir-Hossein Mahmoudi5 , Sharam Samiei6 , Mohammad Ismail Zibaii7

  1. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
  2. Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
  3. Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  4. Department of Physiology and Pharmacology, Pasteur Institute of Iran (IPI), Tehran, Iran.
  5. Ophthalmic Research Center, Research Institute for Ophthalmology and Vision Science, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  6. Blood Transfusion Research Center High Institute for Research and Education in Transfusion Medicine, Tehran, Iran.
  7. Center for Laser and Plasma Research, Shahid Beheshti University, Tehran, Iran.

Abstract: Hereditary retinal degenerative disorders such as retinitis pigmentosa (RP) are among the leading causes of blindness worldwide. Optogenetics gene therapy introduces light-sensitive proteins into the live retinal cells. Opto-mGluR6 is a chimeric protein consisting of the intracellular domain of the ON-bipolar cell specific receptor, mGluR6, and the light-sensing domain of melanopsin. Melanopsin, is a blue-light-sensitive retinal photopigment (mouse λ = 467 nm). Stimulation with blue light bears the high risk of photochemical damage in the sensory retina. We aimed to design engineered red Opto-mGluR6 with broader, red-shifted action spectrum compared to Opto-mGluR6.

Methods: Three red Opto-mGluR6 were designed through bioinformatics tools. The synthesized genes were cloned into pAAV-MCS-IRES-EGFP plasmid. HEK-GIRK stable cells were transfected with the constructs using calcium phosphate precipitation method. After 48 hours, expression of the control and red Opto-mGluR6 constructs were assessed by RT-PCR and Immunocytochemistry. Membrane proteins was extracted and absorption spectra of the opsins were determined by spectroscopy. Whole-cell voltage clamp on transfected HEK293-GIRK cells was performed under the laser radiations in different frequencies and powers.

Results: Three red shift constructs, (Red Opto-mGluR6) ROM 19 (λmax = 557-730 nm), ROM18 (λmax = 540-557 nm) and ROM17 (λmax = 520-540 nm), were designed through bioinformatics and advanced tools. Opto-mgluR6, ROM19, ROM18 and ROM17 genes were successfully cloned into the designated AAV-MCS-IRES-EGFP vector. Large scale DNA plasmids for the genes were prepared. HEK-GIRK stable cell line was generated and transfection and expression of optogenetic constructs in the engineered HEK293-GIRK cells were confirmed. 480, 540, 540 and 600 nm were determined as the maximum absorbance for the opto-mgluR6, ROM18, ROM17 and ROM19 species. Patch clamp study represented that red shifted optogenetics constructs activated GIRK channels when induced by the designated light. They induced depolarization with effective cation current into transfected HEK-GIRK cell line-Opto-mgluR6 (2020 pA), Rom19 (2629 pA), Rom18 (1436 pA) and ROM17 (2100 pA) at 10 mW power and 5 Hz frequency.

Conclusion: This research revealed that red sifted optogenetics constructs directly coupled light stimuli to G-protein signaling.





اخبــار



برگزار کنندگان کنگره


حامیان کنگره