Determination of human FaFg of polyphenols using allometric scaling.

Certain polyphenols exhibit low permeability; precise prediction of their intestinal absorption is important for understanding internal exposure in humans. Intestinal availability, which represents the fraction of administered compounds that reach the portal blood (FaFg), is calculated by dividing bioavailability (F) by hepatic availability (Fh), and F is obtained from pharmacokinetic data from both intravenous (i.v.) and oral (p.o.) administration. However, human FaFg of polyphenols is hardly reported, as human i.v. data are extremely scarce. In this study, we developed an estimation method for FaFg of polyphenols in humans based on the extrapolation of rat clearance using allometric scaling (allometric scaling-based FaFg calculation method, AS- FaFgCM). First, for quercetin, for which human i.v. data have been reported, we compared the FaFg obtained by AS-FaFgCM with the traditional approach using human i.v. and p.o. data. Less than two-fold difference in FaFg values was observed between the two approaches. Next, we obtained FaFg of structurally diverse polyphenols (genistein, baicalein, resveratrol, and epicatechin) using AS-FaFgCM, demonstrating that all of them were poorly absorbable. Furthermore, to utilize the pharmacokinetic data of the total concentration, including aglycones and metabolites, we modified the AS-FaFgCM to focus on their excretion. The FaFg value of naringenin was obtained using modified AS-FaFgCM and was nearly equal to that of baicalein, a structural isomer of naringenin. This study provides quantitative information on the intestinal absorption of polyphenols using comprehensive estimation methods.

Proton-transporting heliorhodopsins from marine giant viruses.

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.

TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

In many rhodopsins, the retinal Schiff base pKa remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10-5 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10-5 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all-trans to 13-cis photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.

Novel optogenetics tool: Gt_CCR4, a light-gated cation channel with high reactivity to weak light.

Optogenetics is a growing technique which allows manipulation of biological events simply by illumination. The technique is appreciated especially in the neuroscience field because of its availability in controlling neuronal functions. A light-gated cation channel, Cr_ChR2 from Chlamydomonas reinhardtii, is the first and mostly applied to optogenetics for activating neuronal excitability. In addition, the molecular mechanism of Cr_ChR2 has been intensively studied by electrophysiology, spectroscopy, X-ray structural studies, etc. Novel cation channelrhodopsins from Guillardia theta, namely, Gt_CCR1-4, were discovered in 2016 and 2017. These channelrhodopsins are more homologous to haloarchaeal rhodopsins, particularly the proton pumps. Thus these cryptophyte-type light-gated cation channels are structurally and mechanistically distinct from chlorophyte channelrhodopsin such as Cr_ChR2. We here compared the photocurrent properties, cation selectivity, and kinetics between well-known Cr_ChR2 and Gt_CCR4. The light sensitivity of Gt_CCR4 is significantly higher than that of Cr_ChR2, while the channel open lifetime is in the same range as that of Cr_ChR2. Gt_CCR4 shows high Na+ selectivity in which the selectivity ratio for Na+ was 37-fold larger than that for Cr_ChR2, which primarily conducts H+. On the other hand, Gt_CCR4 conducted almost no H+ and no Ca2+ under physiological conditions. Other unique features and the applicability of Gt_CCR4 for optogenetics were discussed.