Biology subjects

Ishikita, H.

Publications and source records attributed to Ishikita, H..

6 recordsLinked to original sources

bioRxiv2025

Rhodopsin 7 is indispensable for regulating the firing rates of olfactory sensory neurons in response to extracellular field potential changes in Drosophila melanogaster

Although extracellular field potential changes are commonly observed in the nervous systems, it remains controversial if extracellular electrical activity contributes to neural processing or whether it is an epiphenomenon associated with neural activity. We previously reported that the extracellular field potential change in compound eyes in response to light stimulation induces firing rate changes in olfactory sensory neurons in female Drosophila melanogaster. Through further investigation, we found that the extracellular field potential within the olfactory sensillum is regulated by octopaminergic neurons in response to the light stimulation and that rhodopsin 7 mediates the firing rate changes in the olfactory sensory neurons in response to field potential changes in a light-independent manner. Structural analysis suggests a voltage-dependent gating mechanism for rhodopsin 7 to respond to the field potential change. This study reveals that the nervous system actively controls the field potential in response to sensory input, resulting in alteration of behavioral patterns as well as neural firing patterns in a context-dependent manner. Significance statementAlthough extracellular electrical activity has been recorded to diagnose neuropsychiatric disorders, it remains uncertain how it can be controlled by the nervous system. Moreover, it is difficult to investigate how neurons change their excitability by responding to the change in the extracellular field potential, as synaptic communication interferes in the ability to isolate the function of extracellular electrical activity. We here show that the extracellular field potential within the olfactory sensillum in Drosophila melanogaster is actively regulated by octopaminergic neurons in response to sensory input. We also provide evidence that rhodopsin, a major light sensor protein, mediates responses to extracellular electrical signals, resulting in alternation of behavioral patterns as well as neural firing patterns in a context-dependent manner.

neuroscience↗

bioRxiv2025

Structural basis for color tuning and passive ion conductance in red-shifted pump-fold channelrhodopsin ChR024

ChR024 is a cation-conducting channelrhodopsin with a red-shifted absorption spectrum, recently discovered from a previously unidentified clade through machine-learning-guided gene mining. Due to its unique position in the phylogenetic tree and distinctive spectral properties, it serves as an important model for elucidating rhodopsin evolution and for engineering optogenetic tools with enhanced performance, but its structure and function remain poorly understood. Here, we present cryo-electron microscopy structures of ChR024 in detergent micelles and lipid nanodiscs at resolutions of 3.22 and 2.45 [A], respectively. The structures reveal an architecture strikingly similar to that of ion-pumping rhodopsins, even more so than other pump-like channelrhodopsins such as ChRmine. Structural, theoretical, and spectroscopic analyses uncover a unique color-tuning mechanism, in which charged residues close and distant from the retinal chromophore cooperatively modulate the pKa of the Schiff base counterion and thereby determine the absorption maximum wavelength. Finally, comparative structural analysis of channel- and pump-type rhodopsins, combined with electrophysiology, provides insights into the molecular boundary between these two functional classes and demonstrates the conversion of an outward proton pump into a light-gated channel. Together, these findings illuminate diverse mechanisms of color tuning and functional specification within the rhodopsin family, paving the way for the rational engineering of next-generation optogenetic tools.

biophysics↗

bioRxiv2023

Absence of electron transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes [Dods, R.et al. (2021) Nature 589, 310-314], we investigated time-dependent changes in the energetics of the electron transfer pathway, considering the entire protein environment of the protein structures and titrating the redox active sites in the presence of all fully equilibrated titratable residues. In the dark and charge-separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer active branch (BL and HL) are higher than those in the electron-transfer inactive branch (BM and HM). However, the stabilization of the [PLPM]*+HL*- state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5-ps ([PLPM]*+HL*- state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL*-(saddling mode) is absent in the HL geometry of the original 5-ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures.

biophysics↗

bioRxiv2023

Characterization of Tryptophan Oxidation Affecting D1 Degradation by FtsH in the Photosystem II Quality Control of Chloroplasts

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH. Competing Interest StatementThe authors have no conflict of interest, financial or otherwise, in relation to this study Impact StatementOxidative modification of Tryptophan residues in the reaction center protein D1 may be a key to drive the Photosystem II repair, likely enhancing accessibility of FtsH protease to D1.

plant biology↗

bioRxiv2021

Proton-mediated gating mechanism of anion channelrhodopsin-1

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 [A]) and Glu68 (5.3 [A]) near the protonated Schiff base. Here we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

biochemistry↗

bioRxiv2019

Basicity of N5 in semiquinone enhances the rate of respiratory electron outflow in Shewanella oneidensis MR-1

Extracellular electron transport (EET) occurs in environmental iron-reducing bacteria and is mediated by an outer membrane multi-heme cytochrome complex (Cyts). It has critical implications for global mineral cycling and electrochemical microbial catalysis. The rate of EET mediated by multiple heme redox centers significantly increases in the presence of flavins and quinones. Their electron free energy does not entirely account for the fact that differential effects on EET rate enhancement vary significantly by factors [≥]100. Here, we report on whole-cell electrochemical analysis of Shewanella oneidensis MR-1 using six flavin analogs and four quinones. We demonstrated that protonation of the nitrogen atom at position 5 (N5) of the isoalloxazine ring is essential for electron outflow acceleration as a bound non-covalent cofactor of Cyts. EET mediated by Cyts was accelerated at a rate dependent on pKa(N5). The EET rate largely decreased in response to the addition of deuterated water (D2O), while low concentration of D2O (4 %) had little impact on electron free energy difference of the heme and non-covalent bound cofactors, strongly suggesting that the protonation of N5 limits the rate of EET. Our findings directly link EET kinetics to proton transport reaction via N5 and provide a basis for the development of novel strategies for controlling EET-associated biological reactions.\n\nSignificance statementThe potential of various small molecules such as flavins and quinones to enhance the rate of extracellular electron transport (EET) has been exploited to develop environmental energy conversion systems. Flavins and quinones have similar molecular structures but their abilities to enhance EET vary by >100x in Shewanella oneidensis MR-1. These large differences are inconsistent with conventional models, which rely on redox potentials or diffusion constant of shuttling electron mediators. In this study, we demonstrated that the basicity of the nitrogen atom of the isoalloxazine ring (N5) enhances the rate of electron outflow when a flavin or quinone is a non-covalent cofactor of S. oneidensis MR-1 outer membrane c-type cytochromes.

biochemistry↗