Functional Analysis of the Channel Rhodopsin Genes from the Green Algae of the White Sea Basin

O. V. Karpova; Карпова О. В.; E. N. Vinogradova; Виноградова Е. Н.; A. M. Moisenovich; Мойсенович А. М.; O. B. Pustovit; Пустовит О. Б.; А. А. Ramonova; Рамонова А. А.; D. V. Abramochkin; Абрамочкин Д. В.; E. S. Lobakova; Лобакова Е. С.

doi:10.31857/S0320972524080023

Functional Analysis of the Channel Rhodopsin Genes from the Green Algae of the White Sea Basin

Authors: Karpova O.V.¹, Vinogradova E.N.¹^,2, Moisenovich A.M.¹, Pustovit O.B.¹, Ramonova А.А.¹, Abramochkin D.V.¹, Lobakova E.S.¹
Affiliations:
1. Lomonosov Moscow State University
2. National Research Center “Kurtchatov Institute”
Issue: Vol 89, No 8 (2024)
Pages: 1322-1333
Section: Articles
URL: https://journal-vniispk.ru/0320-9725/article/view/277370
DOI: https://doi.org/10.31857/S0320972524080023
EDN: https://elibrary.ru/KEECUH
ID: 277370

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Abstract

Optogenetics as the method of light-controlled regulation of cellular processes is based on the use of the channel rhodopsins that directly generate photoinduced currents. The largest number of channel rhodopsin genes has been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channel rhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channel rhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Polar Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5′-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1–TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1–TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed, and its parameters correspond to the characteristics of the CCR. The first discovered functional channel rhodopsin of Bracteacoccus has no close CCR homologs and may be of interest as a candidate for optogenetics.

Keywords

channel rhodopsins, green algae, photo-induced current, optogenetics

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Russian Federation, 119991 Moscow

References

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2. Fig. 1. Alignment of the translated amino acid sequences (CLUSTAL Omega) of the Haematococcus CCR gene products and their native transcripts. Hl37CCR, 94 aa; Hl98CCR1, 84 aa; Hl98CCR1-1, 97 aa are fragments of the H. lacustris CCR genes [7]; Hl98CCR2, 350 aa is the native transcript of the 98CCR2 gene; native transcripts are HpChR1 H. pluvialis (lacustris), 677 aa (GenBank: JN596950, [9]); HpChR H. pluvialis (lacustris), 449 aa [11]; HNG2ChR Haematococcus sp. NG2, 350 aa. [11]; HdChR H. droebakensis (GenBank: KF992059, 307 aa [10]); CrChR2 C. reinhardtii, 737 aa (GenBank: AF508966, [3]). Functionally significant amino acid residues (given in CrChR2 numbering) are highlighted in color: Glu (82, 83, 90, 97, 101 and 123) – red; Glu235 and Ser245 – blue; Lys257 – green; Ser321 – yellow.

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3. Fig. 2. Alignment of translated amino acid sequences (CLUSTAL Omega) of the products of native CCR transcripts of Bracteacoccus and Chlamydomonas. Ba34CCR, 325 aa is a partial transcript of the 34CCR gene of B. aggregatus NAMSU-BM-5/15; native transcripts are CrChR2 of C. reinhardtii, 737 aa (GenBank: AF508966, [3]); CaChR1 of C. augustae, 715 aa (GenBank: JN596951, [9]); CraChR2 of C. raudensis, 635 aa (GenBank: JN596949, [9]); CyChR1 of C. yellowstonensis, 717 aa. (GenBank: JN596948, [9]). Functionally significant amino acid residues (given in CrChR2 numbering) are highlighted in color: Glu (82, 83, 90, 97, 101, and 123) in red; Glu235 and Ser245 in blue; Lys257 in green; Ser321 in yellow.

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4. Fig. 3. 3D models of identified channelrhodopsins (SWISSMODEL) Ba34CCR (a) and Hl98CCR2 (b). The template is 6eid.1.A, the crystal structure of CrChR2 from C. reinhardtii. N and C indicate the localization of the ends of the polypeptide chain. The color code (from red to blue) shows the level of homology with the template.

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5. Fig. 4. Study of the localization of expressed rhodopsins Ba34CCR and Hl98CCR2 in CHO-K1 cells by the method of intravital confocal microscopy: Ba34CCR-YFP (upper row) and Hl98CCR2-YFP (lower row). Cell membranes are stained with CellBrite Red, nuclei are stained with Hoechst 33342. The scale is 10 μm.

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6. Fig. 5. Characteristics of currents generated by Ba34CCR and Hl98CCR2 proteins upon expression in CHO-K1 cells. a – Representative example of original recordings of current through Hl98CCR2 (I98) induced by switching on light with a wavelength of 490 nm of different intensities at a potential level of –20 mV. b – Representative example of original recordings of current through Ba34CCR (I34) of different intensities induced by switching on light with a wavelength of 490 nm (intensity is indicated at the peak of each curve as a percentage of the maximum and in absolute values of mW/mm2) at a potential level of –20 mV. c – Dependence of peak current I34 and plateau current I34 on the intensity of light of 490 nm. g – Volt-ampere curves of peak current and steady-state current (plateau) I34 induced by 490 nm light at 50% intensity of maximum (6.25 mW/mm2)

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