scholarly journals Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Oleg A. Sineshchekov ◽  
Elena G. Govorunova ◽  
Hai Li ◽  
Yumei Wang ◽  
Michael Melkonian ◽  
...  
Keyword(s):  
Green Algae ◽  
Molecular Mechanisms ◽  
Flash Photolysis ◽  
Continuous Light ◽  
Proton Donor ◽  
Continuous Illumination ◽  
Proton Pumps ◽  
Cation Conductance ◽  
Vis Spectroscopy ◽  
Rapid Accumulation

ABSTRACT Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

scholarly journals Conductance mechanisms of rapidly desensitizing cation channelrhodopsins from cryptophyte algae

2020 ◽  
Author(s):  
Oleg A. Sineshchekov ◽  
Elena G. Govorunova ◽  
Hai Li ◽  
Yumei Wang ◽  
Michael Melkonian ◽  
...  
Keyword(s):  
Green Algae ◽  
Molecular Mechanisms ◽  
Flash Photolysis ◽  
Continuous Light ◽  
Proton Donor ◽  
Continuous Illumination ◽  
Proton Pumps ◽  
Cation Conductance ◽  
Vis Spectroscopy ◽  
Rapid Accumulation

ABSTRACTChannelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, BCCR sequences match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs, which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption and Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCECation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-vis spectroscopy and photoelectrophysiology reveal the desensitization is caused by rapid accumulation of a non-conductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

scholarly journals Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Stephan Hirschi ◽  
David Kalbermatter ◽  
Zöhre Ucurum ◽  
Thomas Lemmin ◽  
Dimitrios Fotiadis
Keyword(s):  
Proton Translocation ◽  
Functional Characterization ◽  
Green Light ◽  
Proton Donor ◽  
Molecular Organization ◽  
Primary Proton ◽  
Proton Pumps ◽  
Model Protein ◽  
Dynamics Simulations ◽  
And Function

AbstractThe green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

scholarly journals Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gillian E. Conway ◽  
Zhonglei He ◽  
Ana Lacramioara Hutanu ◽  
George Paul Cribaro ◽  
Eline Manaloto ◽  
...  
Keyword(s):  
Cell Death ◽  
Glioblastoma Multiforme ◽  
Plasma Treatment ◽  
Acridine Orange ◽  
Molecular Mechanisms ◽  
Confocal Imaging ◽  
Atmospheric Plasma ◽  
Cold Atmospheric Plasma ◽  
Rapid Accumulation ◽  
Lysosomal Accumulation

Abstract Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

scholarly journals A New Group of Eubacterial Light-Driven Proton Pumps Lacking the Carboxylic Proton Donor

2016 ◽  
Vol 110 (3) ◽  
pp. 313a
Author(s):  
Andrew Harris ◽  
Milena Ljumovic ◽  
Ana-Nicoleta Bondar ◽  
Yohei Shibata ◽  
Yuto Suzuki ◽  
...  
Keyword(s):  
Proton Donor ◽  
Proton Pumps

Photosynthetic downregulation in the conifer Metasequoia glyptostroboides growing under continuous light: the significance of carbohydrate sinks and paleoecophysiological implications

2006 ◽  
Vol 84 (9) ◽  
pp. 1453-1461 ◽  
Author(s):  
M. Alejandra Equiza ◽  
Michael E. Day ◽  
Richard Jagels ◽  
Xiaochun Li
Keyword(s):  
Photosynthetic Capacity ◽  
Carbon Sink ◽  
Continuous Light ◽  
Continuous Illumination ◽  
Temperate Climate ◽  
Moderate Intensity ◽  
Sink Strength ◽  
Metasequoia Glyptostroboides ◽  
Whole Tree ◽  
Photosynthetic Downregulation

During the Eocene (ca. 45 Ma) a temperate climate at high northern latitudes provided an environment unlike any that currently exists on Earth. The growing season was characterized by long (up to 4 months) periods of continuous, low- to moderate-intensity illumination. While this remarkable light regime offered opportunities for substantial growth, it also imposed physiological challenges consequential to potential carbon sink–source imbalance and resulting downregulation of photosynthetic capacity. To better understand the physiology of adaptation to a continuous-light (CL) environment, we experimentally investigated the effects of CL and carbon sink–source relationships in the deciduous conifer Metasequoia glyptostroboides Hu et Cheng, an extant representative of a genus that was the dominant tree component of many Eocene high-latitude forests. We tested the importance of branch-level and whole-plant sinks in curtailing feedback inhibition and the specific roles of starch and sugars in that process using manipulative experiments. Trees growing under either normal day–night cycles or continuous illumination were subjected to reduction of local, branch-level sinks or both local and whole-tree sinks. Reduction in sink strength led to downregulation of photosynthetic capacity, as evidenced by reduction of photosynthetic rates, carboxylation capacity, and electron transfer capacity. Our results suggest that photosynthetic downregulation is minimized by maintenance of both whole-tree sinks and local sinks. downregulation showed a greater correlation with starch than with sugar content, and ultrastructural evidence indicated that foliar starch accumulated only in chloroplasts, and was accompanied by reduction in functional chloroplast grana, but showed no evidence of physical disruption of thylakoids.

scholarly journals Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Keiichi Kojima ◽  
Susumu Yoshizawa ◽  
Masumi Hasegawa ◽  
Masaki Nakama ◽  
Marie Kurihara ◽  
...  
Keyword(s):  
Schiff Base ◽  
Proton Pump ◽  
Flash Photolysis ◽  
Blue Shift ◽  
Spectroscopic Studies ◽  
Spectral Shift ◽  
Bulk Solution ◽  
Proton Pumps ◽  
Ph Titration ◽  
Retinal Chromophore

AbstractThe photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named ‘schizorhodopsin (SzR)’ has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

scholarly journals Omeprazole Promotes Chloride Exclusion and Induces Salt Tolerance in Greenhouse Basil

Agronomy ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 355 ◽  
Author(s):  
Petronia Carillo ◽  
Pasqualina Woodrow ◽  
Giampaolo Raimondi ◽  
Christophe El-Nakhel ◽  
Antonio Pannico ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Benzimidazole Derivative ◽  
Ion Homeostasis ◽  
Chloride Concentration ◽  
Co2 Assimilation ◽  
Stomatal Resistance ◽  
Bioactive Molecules ◽  
Proton Pumps

The role of small bioactive molecules (<500 Da) in mechanisms improving resource use efficiency in plants under stress conditions draws increasing interest. One such molecule is omeprazole (OMP), a benzimidazole derivative and inhibitor of animal proton pumps shown to improve nitrate uptake and exclusion of toxic ions, especially of chloride from the cytosol of salt-stressed leaves. Currently, OMP was applied as substrate drench at two rates (0 or 10 μM) on hydroponic basil (Ocimum basilicum L. cv. Genovese) grown under decreasing NO3−:Cl− ratio (80:20, 60:40, 40:60, or 20:80). Chloride concentration and stomatal resistance increased while transpiration, net CO2 assimilation rate and beneficial ions (NO3−, PO43−, and SO42−) decreased with reduced NO3−:Cl− ratio under the 0 μM OMP treatment. The negative effects of chloride were not only mitigated by the 10 μM OMP application in all treatments, with the exception of 20:80 NO3−:Cl−, but plant growth at 80:20, 60:40, and 40:60 NO3−:Cl− ratios receiving OMP application showed maximum fresh yield (+13%, 24%, and 22%, respectively), shoot (+10%, 25%, and 21%, respectively) and root (+32%, 76%, and 75%, respectively) biomass compared to the corresponding untreated treatments. OMP was not directly involved in ion homeostasis and compartmentalization of vacuolar or apoplastic chloride. However, it was active in limiting chloride loading into the shoot, as manifested by the lower chloride concentration in the 80:20, 60:40, and 40:60 NO3−:Cl− treatments compared to the respective controls (−41%, −37%, and −24%), favoring instead that of nitrate and potassium while also boosting photosynthetic activity. Despite its unequivocally beneficial effect on plants, the large-scale application of OMP is currently limited by the molecule’s high cost. However, further studies are warranted to unravel the molecular mechanisms of OMP-induced reduction of chloride loading to shoot and improved salt tolerance.

Sign in / Sign up

Export Citation Format

Share Document