Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

[The Position and Roles of Occupational Health and Safety Functions in the UK Risk Management System].

Studying the crisis management systems of other developed countries has been effective in clarifying what the position and roles of occupational health and safety functions should be in the crisis management system in Japan. In addition to previous studies on the US system, we studied the UK system. We gathered relevant information by visiting the headquarters of the Health and Safety Executive (HSE) and the Office for Nuclear Regulation (ONR), as well as obtaining information available via the internet. In the UK, the crisis management system was developed based on the Civil Contingencies Act of 2004, which regulated the system of planning and actual correspondence. The system was established with a framework that is consistent at both the national and local levels regardless of the type of crisis. The HSE has health and safety experts involved in all aspects and levels of the crisis management system. The HSE and ONR also assist in emergency planning for serious accidents involving chemical and radioactive substances. We observed the following three common features for ensuring the health and safety of crisis responders in the US and UK: 1) establishing an all-hazard model, a common crisis response system for all types of crisis, 2) ensuring functions to protect the health and safety of workers in a crisis at the national and local levels, 3) ensuring provision of expertise on health and safety for effective management during a crisis. These findings should be used to improve the system in Japan.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

Blocking effect of NIP-142 on the KCNQ1/KCNE1 channel current expressed in HEK293 cells.

We examined the effect of NIP-142, a benzopyran compound with terminating effect on experimental atrial arrhythmia, on the KCNQ1/KCNE1 channel, which underlies the slow component of the cardiac delayed rectifier potassium channel (I(Ks)). NIP-142, as well as chromanol 293B, showed concentration-dependent blockade of the current expressed in HEK293 cells; the EC(50) value of NIP-142 and chromanol 293B for the inhibition of tail current was 13.2 µM and 4.9 µM, respectively. These results indicate that NIP-142 has blocking effect on the KCNQ1/KCNE1 channel current.

Correct disulfide pairing is required for the biological activity of crustacean androgenic gland hormone (AGH): synthetic studies of AGH.

Androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, is a heterodimeric glycopeptide. In this study, we synthesized AGH with a homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of a semisynthetic peptide differed from that of a recombinant peptide prepared in a baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with a GlcNAc moiety was chemically synthesized by the selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.

Differentiation of photocycle characteristics of flavin-binding BLUF domains of α- and ß-subunits of photoactivated adenylyl cyclase of Euglena gracilis.

Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACß), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACßF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACßF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACßF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACßF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACßF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACßF2 plays a main role in suppressing the quantum efficiency.

Acute impact of right ventricular infarction on early hemodynamic course after inferior myocardial infarction.

BACKGROUND: Right ventricular myocardial infarction (RVMI) is the major cause of hypotension and/or shock (HpS) after acute inferior myocardial infarction (inferior AMI). It is, however, unclear how RVMI affects the acute hemodynamic course. METHODS AND RESULTS: In the present study, 153 patients with inferior AMI caused by right coronary artery occlusion were examined. Associations between in-hospital outcome and HpS before admission (preER-HpS) or HpS after admission (postER-HpS) were assessed using multivariate logistic regression analysis. Multivariate analysis was also conducted to determine a predictor for postER-HpS, including clinical findings in the emergency room as independent variables. HpS developed in 48.4% of patients with inferior AMI. Patients with RVMI more frequently had HpS than their counterparts in the first 6 h after infarction onset. RVMI was, however, not associated with preER-HpS, but was independently with postER-HpS (odds ratio (OR): 10.1; 4.0-27.7), whereas left ventricular failure was associated with preER-HpS, but not with postER-HpS. Furthermore, RVMI (OR: 9.4; 3.6-27.1) identified at presentation predicted postER-HpS. CONCLUSIONS: Independent of concomitant left ventricular involvement, RVMI was significantly associated with postER-HpS, but not with preER-HpS. These findings highlight the importance of identifying RVMI immediately after admission in the setting of inferior AMI. (Circ J 2010; 74: 148 - 155).

Protein phosphatase type 1-interacting protein Ysw1 is involved in proper septin organization and prospore membrane formation during sporulation.

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Delta mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Delta cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.

Structural study of single-walled carbon nanotube films doped by a solution method.

Herein, we have studied a structure of SWNT films doped by organic molecules. In this study, we reacted organic molecules with SWNT films by a vapor phase and a liquid phase, respectively. The structure of doped SWNT films was investigated using synchrotron X-ray powder diffraction measurements, and we found the clear difference between the vapor phase reaction (v-doped) and the liquid phase reaction (I-doped). In v-doped SWNT films, organic molecules are predominantly encapsulated inside SWNTs, although molecules adsorbed on the surface of SWNT bundles in l-doped SWNT films.

Identification and characterization of transcriptional control region of the human beta 1,4-mannosyltransferase gene.

All asparagine-linked glycans (N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT-1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (ß1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT-1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1 kb region upstream from HMT-1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from -33 to -1 positions might act in HMT-1 transcription at basal level and that the region from -200 to -42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200 bp region upstream from HMT-1 ORF might significantly upregulate HMT-1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at -148 to -134 positions should serve as a strong silencer. The control of the HMT-1 transcription by these motifs resided within the 200 bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT-1 expression.

Dynamic localization of a yeast development-specific PP1 complex during prospore membrane formation is dependent on multiple localization signals and complex formation.

During the developmental process of sporulation in Saccharomyces cerevisiae, membrane structures called prospore membranes are formed de novo, expand, extend, acquire a round shape, and finally become plasma membranes of the spores. GIP1 encodes a regulatory/targeting subunit of protein phosphatase type 1 that is required for sporulation. Gip1 recruits the catalytic subunit Glc7 to septin structures that form along the prospore membrane; however, the molecular basis of its localization and function is not fully understood. Here we show that Gip1 changes its localization dynamically and is required for prospore membrane extension. Gip1 first associates with the spindle pole body as the prospore membrane forms, moves onto the prospore membrane and then to the septins as the membrane extends, distributes around the prospore membrane after closure, and finally translocates into the nucleus in the maturing spore. Deletion and mutation analyses reveal distinct sequences in Gip1 that are required for different localizations and for association with Glc7. Binding to Glc7 is also required for proper localization. Strikingly, localization to the prospore membrane, but not association with septins, is important for Gip1 function. Further, our genetic analysis suggests that a Gip1-Glc7 phosphatase complex regulates prospore membrane extension in parallel to the previously reported Vps13, Spo71, Spo73 pathway.

The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy.

Involvement of glutamate 97 in ion influx through photo-activated channelrhodopsin-2.

The light absorption of a channelrhodopsin-2 (ChR2) is followed by conformational changes to the molecule, which allows the channel structure to become permeable to cations. Previously, a single point mutation in ChR2, which replaces glutamate residue 97 with a nonpolar alanine (E97A), was found to attenuate the photocurrent, suggesting that the E97 residue is involved in ion flux regulation. Here, the significance of E97 and its counterpart ChR1 (E136) were extensively studied by mutagenesis, whereby we replaced these glutamates with aspartate (D), glutamine (Q) or arginine (R). We found that the charge at this position strongly influences ion permeation and that the photocurrents were attenuated in the order of ChR2>E97D≈E97Q>E97R. We observed similar results with our chimeric/synthetic/artificial construct, ChR-wide receiver (ChRWR), which contains the first to fifth transmembrane helices of ChR1. The E-to-Q or E-to-R mutations, but not the E-to-D mutation, strongly retarded the sensitivity to the Gd(3+)-dependent blocking of the ChR1 or ChR2 channels. Our results suggest that the glutamate residue at this position lies in the outer pore, where it interacts with a cation to facilitate dehydration, and that this residue is the primary binding target of Gd(3+).

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Determination of dioxin concentrations in fish and seafood samples using a highly sensitive reporter cell line, DR-EcoScreen cells.

There is a strong need for the development of relatively rapid and low-cost bioassays for the determination of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) in environmental and food samples. In this study, we applied a reporter gene assay using DR-EcoScreen cells (DR-cell assay), which is highly sensitive to dioxins, to the determination of PCDD/Fs and dl-PCBs in fish and seafood samples. The PCDD/Fs and dl-PCBs were extracted from homogenated samples (10 g) of 30 fish and shellfish, purified by clean-up procedure using a multilayered silica gel column and an alumina column, and applied to DR-cell assay. Interestingly, the bioanalytical equivalent (BEQ) values obtained from the DR-cell assay [<0.1∼5.4 pg BEQ g(-1) wet weight (ww)] were closely correlated with the toxicity equivalent (TEQ) values from conventional high-resolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS) analysis (r(2)=0.912), and the slope of regression line was 0.913. Therefore, we multiplied the BEQ values from the DR-cell assay by a conversion coefficient (1.095, the reciprocal of 0.913) to approximate the TEQ values from the HRGC-HRMS analysis. Furthermore, we used this DR-cell assay to perform a prescreening test of PCDD/Fs and dl-PCBs in 16 fish and seafood samples purchased from a supermarket, revealing that a sample from the fatty flesh of a bluefin tuna exceeded 8 pg TEQ g(-1)ww (the European Union-tolerance limit). Taken together, these results suggest that the DR-cell assay might be applicable as a rapid and low-cost prescreening method to determine dioxin levels in fish and seafood samples.

Comparative study on the nuclear hormone receptor activity of various phytochemicals and their metabolites by reporter gene assays using Chinese hamster ovary cells.

Phytochemicals are naturally present in a wide variety of plants, and have been suggested to exert a number of effects beneficial to human health. Several phytochemicals possess estrogenic activity through estrogen receptor alpha (ERalpha) and ERbeta, and are, therefore, termed phytoestrogens. In this study, we examined whether various phytochemicals have agonistic and/or antagonistic activity against six human nuclear receptors (ERalpha, ERbeta, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor alpha(1) (TRalpha(1)) and TRbeta(1)) by in vitro reporter gene assays using Chinese hamster ovary cells. Of the 31 phytochemicals tested, including flavonoids, isoflavonoids, coumestan, lignans, catechins and their metabolites, 20 compounds showed estrogenic activity via ERalpha and/or ERbeta, and we ranked these phytochemicals according to their estrogenic potency via ERalpha and ERbeta. As a result, coumestrol and genistein strongly activated ERalpha and ERbeta at very low concentrations of <1x10(-10) M. Most phytochemicals showing estrogenic activity also exhibited agonistic activity against ERbeta at lower concentrations than those for ERalpha, and two typical isoflavones, genistein and daidzein, in particular, showed a potent preference for ERbeta. Further, we found that baicalein has ERbeta antagonistic activity, and two compounds, enterolacton and O-desmethylangolensin, have AR antagonistic activity. Nevertheless, none of tested compounds showed AR agonistic activity together with GR, TRalpha(1) and TRbeta(1) agonistic/antagonistic activity. These results suggest that various phytochemicals or their metabolites preferentially interact with ERalpha/beta among the six nuclear hormone receptors tested, and that the ERbeta agonistic activity, in particular, of these compounds may be associated with various beneficial effects on human health.

Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas.

A light signal is converted into an electrical one in a single molecule named channelrhodopsin, one of the archaea-type rhodopsins in unicellular green algae. Although highly homologous, two molecules of this family, channelrhodopsin-1 (ChR1) and -2 (ChR2), are distinct in photocurrent properties such as the wavelength sensitivity, desensitization, and turning-on and -off kinetics. However, the structures regulating these properties have not been completely identified. Photocurrents were analyzed for several chimera molecules made by replacing N-terminal segments of ChR2 with the homologous counterparts of ChR1. We found that the wavelength sensitivity of the photocurrent was red-shifted with negligible desensitization and slowed turning-on and -off kinetics when replacement was made with the segment containing the fifth transmembrane helix of ChR1. Therefore, this segment is involved in the determination of photocurrent properties, the wavelength sensitivity, and the kinetics characterizing ChR1 and ChR2. Eight amino acid residues differentiating this segment were exchanged one-by-one, and the photocurrent properties of each targeted mutant ChR2 were further analyzed. Among them, position Tyr(226)(ChR1)/Asn(187)(ChR2) is one of the molecular determinants involved in the wavelength sensitivity, desensitization, and turning-on and -off kinetics. It is suggested that these amino acid residues directly or indirectly interact with the chromophore as well as with the protein structure determining the photocurrent kinetics. Some of the chimera channelrhodopsins are suggested to have several advantages over the wild-type ChR2 in the introduction of light-induced membrane depolarization for the purpose of artificial stimulation of neurons in vivo and visual prosthesis for photoreceptor degeneration.

Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2.

Channelrhodopsin-2 (ChR2), one of the algal light-gated cation channel rhodopsins, contains five peculiar glutamic acid residues in the N-terminal region corresponding to the second to third transmembrane helices. Here we made systematic mutations of these polar amino acid residues of ChR2 into nonpolar alanine, and evaluated their photocurrent properties. Amongst them, the photocurrent generated by the E97A mutation, ChR2(E97A), was much smaller than expected from its expression. The ChR2(E97A) photocurrent was similar to wild-type ChR2 in the kinetic profiles, the reversal potential and the dependency to the light power density. Our results suggest that the residue E97 is one of the molecular determinants involved in the ion flux regulation.

High current density in light-emitting transistors of organic single crystals.

We measured the external electroluminescence quantum efficiency (eta(ext)) in light-emitting field-effect transistors (LETs) made of organic single crystals and found that, in the ambipolar transport region, eta(ext) is not degraded up to several hundreds A/cm(2) current-density range, which is 2 orders of magnitude larger than that achieved in conventional organic light-emitting diodes. The present result indicates the single-crystal organic LET is a promising device structure that is free from various kinds of nonradiative losses such as exciton dissociation near electrodes and exciton annihilations.