reporter. Using the second methods we found out that the UCS domain but not the central domain pr... more reporter. Using the second methods we found out that the UCS domain but not the central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS domain and the central domain bind the myosin head, only the UCS domain displays chaperone activity. Funded by a grant from the American Heart Association (AHA 13GRNT17290006).
Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools f... more Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kineti...
Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of... more Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of optogenetics, where light-driven ion pumps are used for cell hyperpolarization and voltage sensing. We identified an opsin-encoding gene (CsR) in the genome of the arctic alga Coccomyxa subellipsoidea C-169 that can produce large photocurrents in Xenopus oocytes. We used this property to analyze the function of individual residues in proton pumping. Modification of the highly conserved proton shuttling residue R83 or its interaction partner Y57 strongly reduced pumping power. Moreover, this mutation converted CsR at moderate electrochemical load into an operational proton channel with inward or outward rectification depending on the amino acid substitution. Together with molecular dynamics simulations, these data demonstrate that CsR-R83 and its interacting partner Y57 in conjunction with water molecules forms a proton shuttle that blocks passive proton flux during the dark-state but promotes proton movement uphill upon illumination. Microbial rhodopsins, which are widely distributed across archaea, bacteria, and some eukarya branches, are categorized as sensory rhodopsins, ion channels, and ion pumps 1,2. They are light-activated, seven-transmembrane proteins with an all-trans retinal as a chromophore. Photon absorption redistributes electrons within the protonated retinal Schiff base that promotes isomerization around the 13-cis bond followed by a cyclic cascade of proton transfer, water redistribution, and conformational changes within the protein that may be monitored by UV/Vis and infrared spectroscopy 3-5. In the context of optogenetics, outward directed proton pumps, as well as inward directed chloride pumps, can be utilized as neural silencers or fluorescence-based voltage sensors 6-8. The function of all microbial proton pumps within the native environment or when applied experimentally depends absolutely on their performance at moderate to high electrochemical load. Surprisingly, our knowledge about the contribution of individual residues to proton pump strength remains very limited despite extensive research on bacteriorhodopsin (BR) since 1971. These studies have mostly been conducted by spectroscopic methods using BR in native purple membranes or BR mutants in detergent at zero voltage. Only the voltage dependence of a few pumps has been determined by electrical measurements of Xenopus laevis oocytes; however, the photocurrent amplitudes in these experiments were too small for more comprehensive studies 9-13. Therefore, various conflicting issues regarding the transport mechanism of proton pumps remained unresolved, including the origin of the proton driving force, the background for voltage dependence of the photocycle, and the functional importance of the non-conserved proton release group. Here we report our characterization of Coccomyxa Rhodopsin (abbreviated as CsR), a proton-pumping rhodopsin from the unicellular arctic freshwater alga Coccomyxa subellipsoidea C-169. This psychrotolerant alga resides in temperatures ranging from − 50 °C to + 25 °C 14. CsR produces huge photocurrents in
Background: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling c... more Background: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3-phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction.
Microbial or type-1 rhodopsins are light sensitive proteins that utilize all-trans retinal as chr... more Microbial or type-1 rhodopsins are light sensitive proteins that utilize all-trans retinal as chromophore. Microbial rhodopsins are present in archaea, eubacteria and eukaryotes. Their broad and patchy distribution among the three domains of life is attributed to the lateral gene transfer mechanism of evolution. Microbial rhodopsins function as sensory rhodopsins, light-gated ion pumps and light-activated ion channels in nature. In this review, we present functional diversity and optogenetics applications of microbial rhodopsins.
Blue light sensors using FAD (BLUF) domains are flavin based blue light photoreceptors. The BLUF ... more Blue light sensors using FAD (BLUF) domains are flavin based blue light photoreceptors. The BLUF domains are often fused with various effector domains. BLUF domain coupled with adenylyl cyclase domain is known as photoactivated adenylyl cyclase (PAC). Naegleria gruberi genome database analysis revealed the presence of four PACs. Each of the photoactivated adenylyl cyclases from Naegleria gruberi (NgPACs) is composed of a BLUF domain and an adenylyl cyclase domain. Light regulated enzymatic activity of recombinant NgPAC1 protein was assayed in dark and after blue light irradiation by measuring the cAMP level. Experimental results showed that the NgPAC1 protein exhibits light regulated cyclase activity. In this report, we have also demonstrated that the recombinant NgPAC1 exits as an oligomer in solution.
GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular ... more GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADPribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch-off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross-linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20-kDa monomeric and 40-kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.
Inherited mutations of SCN5A, the gene that encodes Na V 1.5, the alpha subunit of the principle ... more Inherited mutations of SCN5A, the gene that encodes Na V 1.5, the alpha subunit of the principle voltage-gated Na + channel in the heart, cause congenital Long QT Syndrome variant 3 (LQT-3) by perturbation of channel inactivation. LQT-3 mutations induce small, but aberrant, inward current that prolongs the ventricular action potential and subjects mutation carriers to arrhythmia risk dictated in part by the biophysical consequences of the mutations. Most previously investigated LQT-3 mutations are associated with increased arrhythmia risk during rest or sleep. Here we report a novel LQT-3 mutation discovered in a pediatric proband diagnosed with LQTS but who experienced cardiac events during periods of mild exercise as well as rest. The mutation, which changes a single amino acid (S1904L) in the Na V 1.5 carboxy terminal domain, disrupts the channel inactivation gate complex and promotes late Na + channel currents, not by promoting a bursting mode of gating, but by increasing the propensity of the channel to reopen during prolonged depolarization. Incorporating a modified version of the Markov model of the Na V 1.5 channel into a mathematical model of the human ventricular action potential predicts that the biophysical consequences of the S1904L mutation result in action potential prolongation that is seen for all heart rates but, in contrast to other previously-investigated LQT-3 mutant channels, is most pronounced at fast rates resulting in a drastic reduction in the cells ability to adapt APD to heart rate.
The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic... more The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.
Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-typ... more Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all- trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.
Proceedings of the National Academy of Sciences, 2003
Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represe... more Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii . ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii , were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H + and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracell...
Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the p... more Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.
The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of t... more The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACa and PACb. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.
Journal of Photochemistry and Photobiology A: Chemistry, 2011
The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi ... more The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in
Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. I... more Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D 470 /D 480. Laser flashes converted both with ≈ 200 s into major photointermediates P 510 /P 530 , which reverted back to the dark states with ≈ 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P 500 /P 515 and P 390 were detected on a ns to s time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels. The behavior of microalgae like Chlamydomonas is controlled by primordial eyes. The earliest events that are recorded from these eyes after light excitation are extremely fast photoreceptor currents (1-3) that are carried by Ca 2ϩ and H ϩ under most conditions (3, 4). The currents are generated by channelrhodopsins (ChRs), 4 microbial type rhodopsins with intrinsic ion conductance (5-7). ChRs gain their light sensitivity and gating properties from the covalently bound chromophore alltrans-retinal, which isomerizes after light absorption (8, 9). Studies in Xenopus oocytes and HEK293 cells revealed ChRs to be proton channels, which also conduct cations like Na ϩ , K ϩ , and Ca 2ϩ (5, 6, 10). In these studies, light flashes evoked the ion * This work was supported by Deutsche Forschungsgemeinschaft Grants Sfb
reporter. Using the second methods we found out that the UCS domain but not the central domain pr... more reporter. Using the second methods we found out that the UCS domain but not the central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS domain and the central domain bind the myosin head, only the UCS domain displays chaperone activity. Funded by a grant from the American Heart Association (AHA 13GRNT17290006).
Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools f... more Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kineti...
Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of... more Interest in microbial rhodopsins with ion pumping activity has been revitalized in the context of optogenetics, where light-driven ion pumps are used for cell hyperpolarization and voltage sensing. We identified an opsin-encoding gene (CsR) in the genome of the arctic alga Coccomyxa subellipsoidea C-169 that can produce large photocurrents in Xenopus oocytes. We used this property to analyze the function of individual residues in proton pumping. Modification of the highly conserved proton shuttling residue R83 or its interaction partner Y57 strongly reduced pumping power. Moreover, this mutation converted CsR at moderate electrochemical load into an operational proton channel with inward or outward rectification depending on the amino acid substitution. Together with molecular dynamics simulations, these data demonstrate that CsR-R83 and its interacting partner Y57 in conjunction with water molecules forms a proton shuttle that blocks passive proton flux during the dark-state but promotes proton movement uphill upon illumination. Microbial rhodopsins, which are widely distributed across archaea, bacteria, and some eukarya branches, are categorized as sensory rhodopsins, ion channels, and ion pumps 1,2. They are light-activated, seven-transmembrane proteins with an all-trans retinal as a chromophore. Photon absorption redistributes electrons within the protonated retinal Schiff base that promotes isomerization around the 13-cis bond followed by a cyclic cascade of proton transfer, water redistribution, and conformational changes within the protein that may be monitored by UV/Vis and infrared spectroscopy 3-5. In the context of optogenetics, outward directed proton pumps, as well as inward directed chloride pumps, can be utilized as neural silencers or fluorescence-based voltage sensors 6-8. The function of all microbial proton pumps within the native environment or when applied experimentally depends absolutely on their performance at moderate to high electrochemical load. Surprisingly, our knowledge about the contribution of individual residues to proton pump strength remains very limited despite extensive research on bacteriorhodopsin (BR) since 1971. These studies have mostly been conducted by spectroscopic methods using BR in native purple membranes or BR mutants in detergent at zero voltage. Only the voltage dependence of a few pumps has been determined by electrical measurements of Xenopus laevis oocytes; however, the photocurrent amplitudes in these experiments were too small for more comprehensive studies 9-13. Therefore, various conflicting issues regarding the transport mechanism of proton pumps remained unresolved, including the origin of the proton driving force, the background for voltage dependence of the photocycle, and the functional importance of the non-conserved proton release group. Here we report our characterization of Coccomyxa Rhodopsin (abbreviated as CsR), a proton-pumping rhodopsin from the unicellular arctic freshwater alga Coccomyxa subellipsoidea C-169. This psychrotolerant alga resides in temperatures ranging from − 50 °C to + 25 °C 14. CsR produces huge photocurrents in
Background: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling c... more Background: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3-phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction.
Microbial or type-1 rhodopsins are light sensitive proteins that utilize all-trans retinal as chr... more Microbial or type-1 rhodopsins are light sensitive proteins that utilize all-trans retinal as chromophore. Microbial rhodopsins are present in archaea, eubacteria and eukaryotes. Their broad and patchy distribution among the three domains of life is attributed to the lateral gene transfer mechanism of evolution. Microbial rhodopsins function as sensory rhodopsins, light-gated ion pumps and light-activated ion channels in nature. In this review, we present functional diversity and optogenetics applications of microbial rhodopsins.
Blue light sensors using FAD (BLUF) domains are flavin based blue light photoreceptors. The BLUF ... more Blue light sensors using FAD (BLUF) domains are flavin based blue light photoreceptors. The BLUF domains are often fused with various effector domains. BLUF domain coupled with adenylyl cyclase domain is known as photoactivated adenylyl cyclase (PAC). Naegleria gruberi genome database analysis revealed the presence of four PACs. Each of the photoactivated adenylyl cyclases from Naegleria gruberi (NgPACs) is composed of a BLUF domain and an adenylyl cyclase domain. Light regulated enzymatic activity of recombinant NgPAC1 protein was assayed in dark and after blue light irradiation by measuring the cAMP level. Experimental results showed that the NgPAC1 protein exhibits light regulated cyclase activity. In this report, we have also demonstrated that the recombinant NgPAC1 exits as an oligomer in solution.
GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular ... more GTPases of the Ras superfamily regulate a wide variety of cellular processes including vesicular transport and various secretory pathways of the cell. ADPribosylation factor (ARF) belongs to one of the five major families of the Ras superfamily and serves as an important component of vesicle formation and transport machinery of the cells. The binding of GTP to these Arfs and its subsequent hydrolysis, induces conformational changes in these proteins leading to their enzymatic activities. The dimeric form of Arf is associated with membrane pinch-off during vesicle formation. In this report, we have identified an arf gene from the unicellular green alga Chlamydomonas reinhardtii, CrArf, and showed that the oligomeric state of the protein in C. renhardtii is modulated by the cellular membrane environment of the organism. Protein cross-linking experiments showed that the purified recombinant CrArf has the ability to form a dimer. Both the 20-kDa monomeric and 40-kDa dimeric forms of CrArf were recognized from Chlamydomonas total cell lysate (CrTLC) and purified recombinant CrArf by the CrArf specific antibody. The membranous environment of the cell appeared to facilitate dimerization of the CrArf, as dimeric form was found exclusively associated with the membrane bound organelles. The subcellular localization studies in Chlamydomonas suggested that CrArf mainly localized in the cytosol and was mislocalized in vesicle transport machinery inhibitor treated cells. This research sheds light on the importance of the cellular membrane environment for regulating the oligomeric state of CrArf protein in this organism and associated functional role.
Inherited mutations of SCN5A, the gene that encodes Na V 1.5, the alpha subunit of the principle ... more Inherited mutations of SCN5A, the gene that encodes Na V 1.5, the alpha subunit of the principle voltage-gated Na + channel in the heart, cause congenital Long QT Syndrome variant 3 (LQT-3) by perturbation of channel inactivation. LQT-3 mutations induce small, but aberrant, inward current that prolongs the ventricular action potential and subjects mutation carriers to arrhythmia risk dictated in part by the biophysical consequences of the mutations. Most previously investigated LQT-3 mutations are associated with increased arrhythmia risk during rest or sleep. Here we report a novel LQT-3 mutation discovered in a pediatric proband diagnosed with LQTS but who experienced cardiac events during periods of mild exercise as well as rest. The mutation, which changes a single amino acid (S1904L) in the Na V 1.5 carboxy terminal domain, disrupts the channel inactivation gate complex and promotes late Na + channel currents, not by promoting a bursting mode of gating, but by increasing the propensity of the channel to reopen during prolonged depolarization. Incorporating a modified version of the Markov model of the Na V 1.5 channel into a mathematical model of the human ventricular action potential predicts that the biophysical consequences of the S1904L mutation result in action potential prolongation that is seen for all heart rates but, in contrast to other previously-investigated LQT-3 mutant channels, is most pronounced at fast rates resulting in a drastic reduction in the cells ability to adapt APD to heart rate.
The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic... more The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity-dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase.
Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-typ... more Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all- trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.
Proceedings of the National Academy of Sciences, 2003
Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represe... more Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii . ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii , were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H + and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracell...
Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the p... more Photosynthetic unicellular algae have a unique visual system. In Chlamydomonas reinhardtii, the pigmented eye comprises the optical system and at least five different rhodopsin photoreceptors. Two of them, the channelrhodopsins, are rhodopsin-ion channel hybrids switched between closed and open states by photoisomerization of the attached retinal chromophore. They promise to become a useful tool for noninvasive control of membrane potential and intracellular ion concentrations.
The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of t... more The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACa and PACb. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.
Journal of Photochemistry and Photobiology A: Chemistry, 2011
The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi ... more The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in
Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. I... more Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D 470 /D 480. Laser flashes converted both with ≈ 200 s into major photointermediates P 510 /P 530 , which reverted back to the dark states with ≈ 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P 500 /P 515 and P 390 were detected on a ns to s time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels. The behavior of microalgae like Chlamydomonas is controlled by primordial eyes. The earliest events that are recorded from these eyes after light excitation are extremely fast photoreceptor currents (1-3) that are carried by Ca 2ϩ and H ϩ under most conditions (3, 4). The currents are generated by channelrhodopsins (ChRs), 4 microbial type rhodopsins with intrinsic ion conductance (5-7). ChRs gain their light sensitivity and gating properties from the covalently bound chromophore alltrans-retinal, which isomerizes after light absorption (8, 9). Studies in Xenopus oocytes and HEK293 cells revealed ChRs to be proton channels, which also conduct cations like Na ϩ , K ϩ , and Ca 2ϩ (5, 6, 10). In these studies, light flashes evoked the ion * This work was supported by Deutsche Forschungsgemeinschaft Grants Sfb
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